Apo-2LI and Apo-3 polypeptides

ABSTRACT

Novel polypeptides, designated Apo-3 and Apo-2L1, involved in apoptosis are provided. Compositions including chimeric molecules, nucleic acids, and antibodies are also provided.

RELATED APPLICATIONS

This is a continuation application of U.S. Ser. No. 11/357,080, filedFeb. 21, 2006, which is a continuation application of U.S. Ser. No.10/112,793 filed Mar. 28, 2002, now abandoned, which is a continuationapplication of U.S. Ser. No. 08/828,683 filed Mar. 31, 1997, now U.S.Pat. No. 6,469,144, which is a continuation-in-part application of U.S.Ser. No. 08/625,328 filed Apr. 1, 1996, now abandoned, and U.S. Ser. No.08/710,802 filed Sep. 23, 1996, now abandoned, the contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the identification,isolation, and recombinant production of novel polypeptides involved inmammalian cell apoptosis. In particular, polypeptides designated hereinas “Apo-3” and certain forms thereof designated herein as “Apo-2LI” aredisclosed. Methods of employing the polypeptides of the invention arealso disclosed.

BACKGROUND OF THE INVENTION

Control of cell numbers in mammals is believed to be determined, inpart, by a balance between cell proliferation and cell death. One formof cell death, sometimes referred to as necrotic cell death, istypically characterized as a pathologic form of cell death resultingfrom some trauma or cellular injury. In contrast, there is another,“physiologic” form of cell death which usually proceeds in an orderly orcontrolled manner. This orderly or controlled form of cell death isoften referred to as “apoptosis” [see, e.g., Barr et al.,Bio/Technology, 12:487-493 (1994)]. Apoptotic cell death naturallyoccurs in many physiological processes, including embryonic developmentand clonal selection in the immune system [Itoh et al., Cell, 66:233-243(1991)]. Decreased levels of apoptotic cell death have been associatedwith a variety of pathological conditions, including cancer, lupus, andherpes virus infection [Thompson, Science, 267:1456-1462 (1995)].Increased levels of apoptotic cell death may be associated with avariety of other pathological conditions, including AIDS, Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis, multiplesclerosis, retinitis pigmentosa, cerebellar degeneration, aplasticanemia, myocardial infarction, stroke, reperfusion injury, andtoxin-induced liver disease [see, Thompson, supra]. Apoptotic cell deathis typically accompanied by one or more characteristic morphological andbiochemical changes in cells, such as condensation of cytoplasm, loss ofplasma membrane microvilli, segmentation of the nucleus, degradation ofchromosomal DNA or loss of mitochondrial function. A variety ofextrinsic and intrinsic signals are believed to trigger or induce suchmorphological and biochemical cellular changes [Raff, Nature,356:397-400 (1992); Steller, Science, 267:1445-1449 (1995); Sachs etal., Blood, 82:15 (1993)]. For instance, they can be triggered byhormonal stimuli, such as glucocorticoid hormones for immaturethymocytes, as well as withdrawal of certain growth factors[Watanabe-Fukunaga et al., Nature, 356:314-317 (1992)]. Also, someidentified oncogenes such as myc, rel, and E1A, and tumor suppressors,like p53, have been reported to have a role in inducing apoptosis.Certain chemotherapy drugs and some forms of radiation have likewisebeen observed to have apoptosis-inducing activity [Thompson, supra].

Various molecules, such as tumor necrosis factor-α (“TNF-α”), tumornecrosis factor-β (“TNF-β” or “lymphotoxin”), CD30 ligand, CD27 ligand,CD40 ligand, OX-40 ligand, 4-1BB ligand, Apo-1 ligand (also referred toas Fas ligand or CD95 ligand), TRAIL, and Apo-2 ligand have beenidentified as members of the tumor necrosis factor (“TNF”) family ofcytokines [See, e.g., Gruss and Dower, Blood, 85:3378-3404 (1995); Wileyet al., Immunity, 3:673-682 (1995); Pitti et al., J. Biol. Chem.,271:12687-12690 (1996)]. Among these molecules, TNF-α, TNF-β, CD30ligand, 4-1BB ligand, Apo-1 ligand, TRAIL, and Apo-2 ligand have beenreported to be involved in apoptotic cell death. Both TNF-α and TNF-βhave been reported to induce apoptotic death in susceptible tumor cells[Schmid et al., Proc. Natl. Acad. Sci., 83:1881 (1986); Dealtry et al.,Eur. J. Immunol., 17:689 (1987)]. Zheng et al. have reported that TNF-αis involved in post-stimulation apoptosis of CD8-positive T cells [Zhenget al., Nature, 377:348-351 (1995)]. Other investigators have reportedthat CD30 ligand may be involved in deletion of self-reactive T cells inthe thymus [Amakawa et al., Cold Spring Harbor Laboratory Symposium onProgrammed Cell Death, Abstr. No. 10, (1995)].

Mutations in the mouse Fas/Apo-1 receptor or ligand genes (called lprand gld, respectively) have been associated with some autoimmunedisorders, indicating that Apo-1 ligand may play a role in regulatingthe clonal deletion of self-reactive lymphocytes in the periphery[Krammer et al., Curr. Op. Immunol., 6:279-289 (1994); Nagata et al.,Science, 267:1449-1456 (1995)]. Apo-1 ligand is also reported to inducepost-stimulation apoptosis in CD4-positive T lymphocytes and in Blymphocytes, and may be involved in the elimination of activatedlymphocytes when their function is no longer needed [Krammer et al.,supra; Nagata et al., supra]. Agonist mouse monoclonal antibodiesspecifically binding to the Apo-1 receptor have been reported to exhibitcell killing activity that is comparable to or similar to that of TNF-α[Yonehara et al., J. Exp. Med., 169:1747-1756 (1989)].

Induction of various cellular responses mediated by such TNF familycytokines is believed to be initiated by their binding to specific cellreceptors. Two distinct TNF receptors of approximately 55-kDa (TNFR1)and 75-kDa (TNFR2) have been identified [Hohman et al., J. Biol. Chem.,264:14927-14934 (1989); Brockhaus et al., Proc. Natl. Acad. Sci.,87:3127-3131 (1990); EP 417,563, published Mar. 20, 1991] and human andmouse cDNAs corresponding to both receptor types have been isolated andcharacterized [Loetscher et al., Cell, 61:351 (1990); Schall et al.,Cell, 61:361 (1990); Smith et al., Science, 248:1019-1023 (1990); Lewiset al., Proc. Natl. Acad. Sci., 88:2830-2834 (1991); Goodwin et al.,Mol. Cell. Biol., 11:3020-3026 (1991)]. Extensive polymorphisms havebeen associated with both TNF receptor genes [see, e.g., Takao et al.,Immunogenetics, 37:199-203 (1993)]. Both TNFRs share the typicalstructure of cell surface receptors including extracellular,transmembrane and intracellular regions. The extracellular portions ofboth receptors are found naturally also as soluble TNF-binding proteins[Nophar, Y. et al., EMBO J., 9:3269 (1990); and Kohno, T. et al., Proc.Natl. Acad. Sci. U.S.A., 87:8331 (1990)]. More recently, the cloning ofrecombinant soluble TNF receptors was reported by Hale et al. [J. Cell.Biochem. Supplement 15F, 1991, p. 113 (P424)].

The extracellular portion of type 1 and type 2 TNFRs (TNFR1 and TNFR2)contains a repetitive amino acid sequence pattern of four cysteine-richdomains (CRDs) designated 1 through 4, starting from the NH₂-terminus.Each CRD is about 40 amino acids long and contains 4 to 6 cysteineresidues at positions which are well conserved [Schall et al., supra;Loetscher et al., supra; Smith et al., supra; Nophar et al., supra;Kohno et al., supra]. In TNFR1, the approximate boundaries of the fourCRDs are as follows: CRD1-amino acids 14 to about 53; CRD2-amino acidsfrom about 54 to about 97; CRD3-amino acids from about 98 to about 138;CRD4-amino acids from about 139 to about 167. In TNFR2, CRD1 includesamino acids 17 to about 54; CRD2-amino acids from about 55 to about 97;CRD3-amino acids from about 98 to about 140; and CRD4-amino acids fromabout 141 to about 179 [Banner et al., Cell, 73:431-435 (1993)]. Thepotential role of the CRDs in ligand binding is also described by Banneret al., supra.

A similar repetitive pattern of CRDs exists in several othercell-surface proteins, including the p75 nerve growth factor receptor(NGFR) [Johnson et al., Cell, 47:545 (1986); Radeke et al., Nature,325:593 (1987)], the B cell antigen CD40 [Stamenkovic et al., EMBO J.,8: 1403 (1989)], the T cell antigen OX40 [Mallet et al., EMBO J., 9:1063(1990)] and the Fas antigen [Yonehara et al., supra and Itoh et al.,supra]. CRDs are also found in the soluble TNFR (sTNFR)-like T2 proteinsof the Shope and myxoma poxviruses [Upton et al., Virology, 160:20-29(1987); Smith et al., Biochem. Biophys. Res. Commun., 176:335 (1991);Upton et al., Virology, 184:370 (1991)]. Optimal alignment of thesesequences indicates that the positions of the cysteine residues are wellconserved. These receptors are sometimes collectively referred to asmembers of the TNF/NGF receptor superfamily. Recent studies on p75NGFRshowed that the deletion of CRD1 [Welcher, A. A. et al., Proc. Natl.Acad. Sci. USA, 88:159-163 (1991)] or a 5-amino acid insertion in thisdomain [Yan, H. and Chao, M. V., J. Biol. Chem., 266:12099-12104 (1991)]had little or no effect on NGF binding [Yan, H. and Chao, M. V., supra].p75 NGFR contains a proline-rich stretch of about 60 amino acids,between its CRD4 and transmembrane region, which is not involved in NGFbinding [Peetre, C. et al., Eur. J. Hematol., 41:414-419 (1988);Seckinger, P. et al., J. Biol. Chem., 264:11966-11973 (1989); Yan, H.and Chao, M. V., supra]. A similar proline-rich region is found in TNFR2but not in TNFR1.

Itoh et al. disclose that the Apo-1 receptor can signal an apoptoticcell death similar to that signaled by the 55-kDa TNFR1 [Itoh et al.,supra]. Expression of the Apo-1 antigen has also been reported to bedown-regulated along with that of TNFR1 when cells are treated witheither TNF-α or anti-Apo-1 mouse monoclonal antibody [Krammer et al.,supra; Nagata et al., supra]. Accordingly, some investigators havehypothesized that cell lines that co-express both Apo-1 and TNFR1receptors may mediate cell killing through common signaling pathways[Id.].

The TNF family ligands identified to date, with the exception oflymphotoxin-α, are type II transmembrane proteins, whose C-terminus isextracellular. In contrast, the receptors in the TNF receptor (TNFR)family identified to date are type I transmembrane proteins. In both theTNF ligand and receptor families, however, homology identified betweenfamily members has been found mainly in the extracellular domain(“ECD”). Several of the TNF family cytokines, including TNF-α, Apo-1ligand and CD40 ligand, are cleaved proteolytically at the cell surface;the resulting protein in each case typically forms a homotrimericmolecule that functions as a soluble cytokine. TNF receptor familyproteins are also usually cleaved proteolytically to release solublereceptor ECDs that can function as inhibitors of the cognate cytokines.

Two of the TNFR family members, TNFR1 and Fas/Apo1 (CD95), can activateapoptotic cell death [Chinnaiyan and Dixit, Current Biology, 6:555-562(1996); Fraser and Evan, Cell; 85:781-784 (1996)]. TNFR1 is also knownto mediate activation of the transcription factor, NF-κB [Tartaglia etal., Cell, 74:845-853 (1993); Hsu et al., Cell, 84:299-308 (1996)]. Inaddition to some ECD homology, these two receptors share homology intheir intracellular domain (ICD) in an oligomerization interface knownas the death domain [Tartaglia et al., supra]. Death domains are alsofound in several metazoan proteins that regulate apoptosis, namely, theDrosophila protein, Reaper, and the mammalian proteins referred to asFADD/MORT1, TRADD, and RIP [Cleaveland and Ihle, Cell, 81:479-482(1995)]. Using the yeast-two hybrid system, Raven et al. report theidentification of protein, wsl-1, which binds to the TNFR1 death domain[Raven et al., Programmed Cell Death Meeting, Sep. 20-24, 1995, Abstractat page 127; Raven et al., European Cytokine Network, 7: Abstr. 82 atpage 210 (April-June 1996)]. The wsl-1 protein is described as beinghomologous to TNFR1 (48% identity) and having a restricted tissuedistribution. According to Raven et al., the tissue distribution ofwsl-1 is significantly different from the TNFR1 binding protein, TRADD.

Upon ligand binding and receptor clustering, TNFR1 and CD95 are believedto recruit FADD into a death-inducing signalling complex. CD95purportedly binds FADD directly, while TNFR1 binds FADD indirectly viaTRADD [Chinnaiyan et al., Cell, 81:505-512 (1995); Boldin et al., J.Biol. Chem., 270:387-391 (1995); Hsu et al., supra; Chinnaiyan et al.,J. Biol. Chem., 271:4961-4965 (1996)]. It has been reported that FADDserves as an adaptor protein which recruits the thiol proteaseMACHα/FLICE into the death signalling complex [Boldin et al., Cell,85:803-815 (1996); Muzio et al., Cell, 85:817-827 (1996)]. MACHα/FLICEappears to be the trigger that sets off a cascade of apoptoticproteases, including the interleukin-1β converting enzyme (ICE) andCPP32/Yama, which may execute some critical aspects of the cell deathprogramme [Fraser and Evan, supra]

It was recently disclosed that programmed cell death involves theactivity of members of a family of cysteine proteases related to the C.elegans cell death gene, ced-3, and to the mammalian IL-1-convertingenzyme, ICE. The activity of the ICE and CPP32/Yama proteases can beinhibited by the product of the cowpox virus gene, cmmA [Ray et al.,Cell, 69:597-604 (1992); Tewari et al., Cell, 81:801-809 (1995)]. Recentstudies show that CrmA can inhibit TNFR1- and CD95-induced cell death[Enari et al., Nature, 375:78-81 (1995); Tewari et al., J. Biol. Chem.,270: 3255-3260 (1995)].

As reviewed recently by Tewari et al., TNFR1, TNFR2 and CD40 modulatethe expression of proinflammatory and costimulatory cytokines, cytokinereceptors, and cell adhesion molecules through activation of thetranscription factor, NF-κB [Tewari et al., Curr. Op. Genet. Develop.,6:39-44 (1996)]. NF-κB is the prototype of a family of dimerictranscription factors whose subunits contain conserved Rel regions[Verma et al., Genes Develop., 9:2723-2735 (1996); Baldwin, Ann. Rev.Immunol., 14:649-681 (1996)]. In its latent form, NF-κB is complexedwith members of the IκB inhibitor family; upon inactivation of the IκBin response to certain stimuli, released NF-κB translocates to thenucleus where it binds to specific DNA sequences and activates genetranscription. TNFR proteins may also regulate the AP-1 transcriptionfactor family [Karin, J. Biol. Chem., 270:16483-16486 (1995)]. AP-1represents a separate family of dimeric transcriptional activatorscomposed of members of the Fos and Jun protein families [Karin, supra].AP-1 activation is believed to be mediated by immediate-early inductionof fos and jun through the mitogen-activated protein kinases ERK and JNK(Jun N-terminal kinase; also known as stress-activated protein kinase,SAPK), as well as by JNK-dependent phosphorylation of Jun proteins[Karin, supra; Kyriakis et al., J. Biol. Chem., 271:24313-24316 (1996)].Transcriptional regulation by TNFR family members is mediated primarilyby members of the TNF receptor associated factor (TRAF) family [Rothe etal., Cell, 78:681-692 (1994); Hsu et al., Cell, 84:299-308 (1996); Liuet al., Cell, 87:565-576 (1996)].

For a review of the TNF family of cytokines and their receptors, seeGruss and Dower, supra.

SUMMARY OF THE INVENTION

Applicants have identified cDNA clones that encode novel polypeptides,designated in the present application as “Apo-3.” The Apo-3 polypeptidehas surprisingly been found to stimulate or induce apoptotic activity inmammalian cells. It is believed that Apo-3 is a member of the TNFRfamily; full-length native sequence human Apo-3 polypeptide exhibitssome similarities to some known TNFRs, including TNFR1 and CD95. Inparticular, full-length native sequence human Apo-3 exhibits similarityto the TNFR family in its extracellular cysteine-rich repeats andresembles TNFR1 and CD95 in that it contains a cytoplasmic death domainsequence.

Applicants have also identified cDNA clones that encode a polypeptide,designated “Apo-2 ligand inhibitor” or “Apo-2LI”. Although not beingbound to any particular theory, it is presently believed that Apo-2LIcomprising amino acid residues 1 to 181 of FIG. 1 (SEQ ID NO:1) [andwhich correspond to amino acid residues 1 to 181 of the sequence of FIG.4 (SEQ ID NO:6)] may be a soluble, truncated or secreted form of Apo-3.

In one embodiment, the invention provides isolated Apo-2LI. Inparticular, the invention provides isolated native sequence Apo-2LI,which in one embodiment, includes an amino acid sequence comprisingresidues 1 to 181 of FIG. 1 (SEQ ID NO:1). In other embodiments, theisolated Apo-2LI comprises one or more cysteine-rich domains of thesequence of FIG. 1, or comprises biologically active polypeptidescomprising at least about 80% identity with native sequence Apo-2LIshown in FIG. 1 (SEQ ID NO:1).

In another embodiment, the invention provides chimeric moleculescomprising Apo-2LI fused to another, heterologous polypeptide or aminoacid sequence. An example of such a chimeric molecule comprises anApo-2LI amino acid sequence fused to an immunoglobulin constant domainsequence.

In another embodiment, the invention provides an isolated nucleicacid-molecule encoding Apo-2LI. In one aspect, the nucleic acid moleculeis RNA or DNA that encodes an Apo-2LI or is complementary to a nucleicacid sequence encoding such Apo-2LI, and remains stably bound to itunder stringent conditions. In one embodiment, the nucleic acid sequenceis selected from:

(a) the coding region of the nucleic acid sequence of FIG. 1 that codesfor residue 1 to residue 181 (i.e., nucleotides 377 through 919; alsoprovided in SEQ ID NO:5), inclusive; or

(b) a sequence corresponding to the sequence of (a) within the scope ofdegeneracy of the genetic code.

In a further embodiment, the invention provides a replicable vectorcomprising the nucleic acid molecule encoding the Apo-2LI operablylinked to control sequences recognized by a host cell transfected ortransformed with the vector. A host cell comprising the vector or thenucleic acid molecule is also provided. A method of producing Apo-2LIwhich comprises culturing a host cell comprising the nucleic acidmolecule and recovering the protein from the host cell culture isfurther provided.

In another embodiment, the invention provides an antibody which binds toApo-2LI.

In another embodiment, the invention provides isolated Apo-3polypeptide. In particular, the invention provides isolated nativesequence Apo-3 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 417 of FIG. 4 (SEQ ID NO:6). Inother embodiments, the isolated Apo-3 polypeptide comprises at leastabout 80% identity with native sequence Apo-3 polypeptide comprisingresidues 1 to 417 of FIG. 4 (SEQ ID NO:6).

In another embodiment, the invention provides an isolated extracellulardomain sequence of Apo-3 polypeptide. The isolated extracellular domainsequence preferably comprises residues 1 to 198 of FIG. 4 (SEQ ID NO:6).

In another embodiment, the invention provides an isolated death domainsequence of Apo-3 polypeptide. The isolated death domain sequencepreferably comprises residues 338 to 417 of FIG. 4 (SEQ ID NO:6).

In another embodiment, the invention provides chimeric moleculescomprising Apo-3 polypeptide fused to a heterologous polypeptide oramino acid sequence. An example of such a chimeric molecule comprises anApo-3 fused to an immunoglobulin sequence. Another example comprises anextracellular domain sequence of Apo-3 fused to a heterologouspolypeptide or amino acid sequence, such as an immunoglobulin sequence.

In another embodiment, the invention provides an isolated nucleic acidmolecule encoding Apo-3 polypeptide. In one aspect, the nucleic acidmolecule is RNA or DNA that encodes an Apo-3 polypeptide or a particulardomain of Apo-3, or is complementary to such encoding nucleic acidsequence, and remains stably bound to it under stringent conditions. Inone embodiment, the nucleic acid sequence is selected from:

(a) the coding region of the nucleic acid sequence of FIG. 4 (SEQ IDNO:9) that codes for residue 1 to residue 417 (i.e., nucleotides 89-91through 1337-1339), inclusive; or

(b) the coding region of the nucleic acid sequence of FIG. 4 (SEQ IDNO:9) that codes for residue 1 to residue 198 (i.e., nucleotides 89-91through 680-682), inclusive;

(c) the coding region of the nucleic acid sequence of FIG. 4 (SEQ IDNO:9) that codes for residue 338 to residue 417 (i.e., nucleotides1100-1102 through 1337-1339), inclusive; or

(d) a sequence corresponding to the sequence of (a), (b) or (c) withinthe scope of degeneracy of the genetic code.

In a further embodiment, the invention provides a vector comprising thenucleic acid molecule encoding the Apo-3 polypeptide or particulardomain of Apo-3. A host cell comprising the vector or the nucleic acidmolecule is also provided. A method of producing Apo-3 is furtherprovided.

In another embodiment, the invention provides an antibody which binds toApo-3.

In another embodiment, the invention provides non-human, transgenic orknock-out animals.

A further embodiment of the invention provides articles of manufactureand kits.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of human Apo-2LI cDNA (SEQ ID NO:5)and its derived amino acid sequence (SEQ ID NO:1).

FIGS. 2A-2B show an alignment of the amino acid sequence encoded byclone 18.1 of Apo-2 ligand inhibitor (amino acids 34-181 of SEQ ID NO:1)with extracellular regions of other members of the human TNF receptorfamily: hTNFR1 (SEQ ID NO:12); hTNFR2 (SEQ ID NO:13); hTNFRrp (SEQ IDNO:14); hFas/Apo1 (SEQ ID NO:15); hLNGFR (SEQ ID NO:16); hCD40 (SEQ IDNO:17); hCD27 (SEQ ID NO:18); hCD30 (SEQ ID NO:19); hOX40 (SEQ IDNO:20).

FIG. 3 shows a silver-stained gel of a protein A purified Apo-2LIimmunoadhesin analyzed under non-reducing (lanes 3-5) or reducing (lanes7-9) conditions.

FIGS. 4A-4C show the nucleotide sequence of native sequence human Apo-3cDNA (SEQ ID NO:9) and its derived amino acid sequence (SEQ ID NO:6).The putative signal sequence and transmembrane domain are underlined,the death domain sequence is boxed, and the potential N-linkedglycosylation sites are marked with an asterisk. Also boxed is thealanine residue which was present in the fetal lung but not in the fetalheart cDNA clone (discussed in Example 4 below).

FIG. 5 shows an alignment and comparison of the ECD sequences of nativesequence human Apo-3 (amino acids 1-197 of SEQ ID NO:9), TNFR1 (SEQ IDNO:21), and Fas/Apo-1/CD95 (SEQ ID NO:22).

FIG. 6 shows an alignment and comparison of the death domain sequencesof native sequence human Apo-3 (amino acids 338-411 of SEQ ID NO:9),TNFR1 (SEQ ID NO:23), Fas/Apo-1/CD95 (SEQ ID NO:24), FADD (SEQ IDNO:25), TRADD (SEQ ID NO:26), RIP (SEQ ID NO:27) and Drosophila Reaper(SEQ ID NO:28).

FIG. 7 shows a schematic alignment of Apo-3, Apo-2LI, TNFR1, andFas/Apo-1. CRD, cysteine-rich domains; TM, transmembrane domain; DD,death domain.

FIG. 8 shows ectopic expression of Apo-3 in HEK 293 cells. Cells weretransfected with pRK5-Apo-3 plus pRK5 (5 μg each) (lane 1); pRK5 alone(10 μg) (lane 2); or pRK5-Apo-3 plus pRK5-CrmA (5 μg each) (lane 3).Cells were metabolically labeled with ³⁵S-Met and ³⁵S-Cys. Cell lysateswere then analyzed by radioimmunoprecipitation using mouse anti-Apo-3antiserum. The molecular weight standards are shown on the left in kDa.

FIGS. 9 a-j illustrate the induction of apoptosis by ectopic expressionof Apo-3 in HEK 293 cells. Apoptosis was examined 36 hours aftertransfection, by morphological analysis (FIGS. 9 a-d); by FACS analysis(FIGS. 9 e-i); and by DNA laddering (FIG. 9 j). Cells were transfectedwith pRK5 alone (10 μg) (FIGS. 9 a; e; j, lane 1); pRK5 plus pRK5-Apo-3(5 μg each) (FIGS. 9 b; f; j, lane 2); pRK5 plus pRK5-CrmA (5 μg each)(FIGS. 9 c; g; j, lane 3); or pRK5-Apo-3 plus pRK5-CrmA (5 μg each)(FIGS. 9 d; h; j, lane 4). Cells in FIGS. 9 a-d were photographed at400× magnification using Hoffmann optics-based light microscopy. Asmeasured by the total number of annexin V-positive cells, the percentapoptosis in FIGS. 9 e-h, respectively, was 37%, 66%, 36% and 26%. Cellsin FIG. 9 i were transfected with the indicated amount of pRK5-Apo-3 orpRK5-TNFR1 and the appropriate amount of pRK5 plasmid to bring the totalamount of DNA to 20 μg.

FIG. 10 shows activation of NF-κB by ectopic expression of Apo-3. HEK293 cells were transfected with 10 μg pRK5 (lanes 1, 4, 7); pRK5-Apo-3(lanes 2, 5, 7); or pRK5-TNFR1 (lanes 3, 6, 9). Nuclear extracts wereprepared 36 hours later and reacted with an irrelevant ³²P-labelledoligonucleotide probe (lanes 1-3); or with a ³²P-labelled NF-κB-specificprobe alone (lanes 4-6) or in the presence of 50-fold excess unlabelledoligonucleotide of the same sequence (lanes 7-9).

FIG. 11 shows activation of Jun N-terminal kinase (JNK) by ectopicexpression of Apo-3. HEK 293 cells were transfected with 10 μg pRK5,pRK5-TNFR1, or pRK5-Apo-3 as indicated. 36 hours later, JNK activity incell extracts was determined using a JNK/SAPK activation kit, whichmeasures JNK activity by analyzing phosphorylation of c-Jun.

FIG. 12 illustrates expression of Apo-3 mRNA in human tissues asdetermined by Northern blot hybridization. In the left hand panel areshown fetal brain (1); lung (2); liver (3); kidney (4). In the righthand panel are shown adult spleen (1); thymus (2); prostate (3); testis(4); ovary (5); small intestine (6); colon (7); and peripheral bloodlymphocytes (8). The sizes of the molecular weight standards are shownon the left in kb.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

The terms “Apo-3 polypeptide” and “Apo-3” when used herein encompassnative sequence Apo-3 and Apo-3 variants (each of which is definedherein). These terms encompass Apo-3 from a variety of mammals,including humans. The Apo-3 may be isolated from a variety of sources,such as from human tissue types or from another source, or prepared byrecombinant or synthetic methods.

A “native sequence Apo-3” comprises a polypeptide having the same aminoacid sequence as an Apo-3 derived from nature. Thus, a native sequenceApo-3 can have the amino acid sequence of naturally-occurring Apo-3 fromany mammal. Such native sequence Apo-3 can be isolated from nature orcan be produced by recombinant or synthetic means. The term “nativesequence Apo-3” specifically encompasses naturally-occurring truncatedor secreted forms of the Apo-3 (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the Apo-3. Anaturally-occurring variant form of the Apo-3 includes an Apo-3 havingan amino acid deletion at residue 236 in the amino acid sequence shownin FIG. 4 (SEQ ID NO:6). In one embodiment of the invention, the nativesequence Apo-3 is a mature or full-length native sequence Apo-3comprising the amino acid sequence of SEQ ID NO:6. The presentdefinition of native sequence Apo-3 excludes known EST sequences, suchas GenBank W71984.

“Apo-3 variant” means a biologically active Apo-3 as defined belowhaving less than 100% sequence identity with Apo-3 having the deducedamino acid sequence shown in FIG. 4 (SEQ ID NO:6) for a full-lengthnative sequence human Apo-3. Such Apo-3 variants include, for instance,Apo-3 polypeptides wherein one or more amino acid residues are added atthe N- or C-terminus of, or within, the sequence of SEQ ID NO:6; fromabout one to 24 amino acid residues are deleted (including a singleamino acid deletion at residue 236 in the amino acid sequence shown inFIG. 4 (SEQ ID NO:6), or optionally substituted by one or more aminoacid residues; and derivatives thereof, wherein an amino acid residuehas been covalently modified so that the resulting product has anon-naturally occurring amino acid. Ordinarily, an Apo-3 variant willhave at least about 80% sequence identity, more preferably at leastabout 90% sequence identity, and even more preferably at least about 95%sequence identity with the sequence of FIG. 4 (SEQ ID NO:6). The presentdefinition of Apo-3 variant excludes known EST sequences, such asGenBank W71984.

The terms “Apo-2 ligand inhibitor polypeptide” and “Apo-2LI” when usedherein encompass native sequence Apo-2LI and Apo-2LI variants (each ofwhich is defined herein). These terms encompass Apo-2LI from a varietyof mammals, including humans. The Apo-2LI may be isolated from a varietyof sources, such as from human tissue types or from another source, orprepared by recombinant or synthetic methods.

A “native sequence Apo-2LI” comprises a polypeptide having the sameamino acid sequence as an Apo-2LI derived from nature. Thus, a nativesequence Apo-2LI can have the amino acid sequence of naturally-occurringApo-2LI from any mammal. Such native sequence Apo-2LI can be isolatedfrom nature or can be produced by recombinant or synthetic means. Theterm “native sequence Apo-2LI” specifically encompassesnaturally-occurring truncated forms, naturally-occurring variant forms(e.g., alternatively spliced forms) and naturally-occurring allelicvariants. In one embodiment of the invention, the native sequenceApo-2LI comprises the amino acid sequence of SEQ ID NO:1. The presentdefinition of native sequence Apo-2LI excludes known EST sequences, suchas GenBank H41522, H46424, H46211, H46374, H46662, H41851, H49675,H22502, H46378 and H19739.

“Apo-2LI variant” means a biologically active Apo-2LI as defined belowhaving less than 100% sequence identity with Apo-2LI having the deducedamino acid sequence shown in FIG. 1 (SEQ ID NO:1). Such Apo-2LI variantsinclude, for instance, Apo-2LI polypeptides wherein one or more aminoacid residues are added at the N- or C-terminus of, or within, thesequence of SEQ ID NO:1; wherein one or more amino acid residues aredeleted, or optionally substituted by one or more amino acid residues;and derivatives thereof, wherein an amino acid residue has beencovalently modified so that the resulting product has a non-naturallyoccurring amino acid. Optionally, the Apo-2LI includes one or morecysteine-rich domains, and preferably includes one or more cysteine-richdomains comprising amino acids 34 to 71, amino acids 72 to 115, aminoacids 116 to 163, or amino acids 164 to 181 of FIG. 1. Ordinarily, anApo-2LI variant will have at least about 80% sequence identity, morepreferably at least about 90% sequence identity, and even morepreferably at least about 95% sequence identity with the sequence ofFIG. 1 (SEQ ID NO:1). The present definition of Apo-2LI variant excludesknown EST sequences, such as GenBank H41522, H46424, H46211, H46374,H46662, H41851, H49675, H22502, H46378 and H19739.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising at least one of the Apo-3 or Apo-2LI polypeptidesdisclosed herein, or a portion thereof, fused to a “tag polypeptide”.The tag polypeptide has enough residues to provide an epitope againstwhich an antibody can be made, yet is short enough such that it does notinterfere with activity of the Apo-3 or Apo-2LI. The tag polypeptidepreferably also is fairly unique so that the antibody does notsubstantially cross-react with other epitopes. Suitable tag polypeptidesgenerally have at least six amino acid residues and usually betweenabout 8 to about 50 amino acid residues (preferably, between about 10 toabout 20 residues).

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the Apo-3 or Apo-2LInatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe nucleic acid. An isolated nucleic acid molecule is other than in theform or setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from the nucleic acid molecule asit exists in natural cells. However, an isolated Apo-3 nucleic acidmolecule, for instance, includes Apo-3 nucleic acid molecules containedin cells that ordinarily express Apo-3 where, for example, the nucleicacid molecule is in a chromosomal location different from that ofnatural cells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “antibody” is used in the broadest sense and specificallycovers single monoclonal antibodies (including agonist, antagonist, andneutralizing antibodies) and antibody compositions with polyepitopicspecificity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen.

The monoclonal antibodies herein include hybrid and recombinantantibodies produced by splicing a variable (including hypervariable)domain of an anti-Apo-3 antibody or anti-Apo-2LI antibody with aconstant domain (e.g. “humanized” antibodies), or a light chain with aheavy chain, or a chain from one species with a chain from anotherspecies, or fusions with heterologous proteins, regardless of species oforigin or immunoglobulin class or subclass designation, as well asantibody fragments (e.g., Fab, F(ab′)₂, and Fv), so long as they exhibitthe desired biological activity. See, e.g. U.S. Pat. No. 4,816,567 andMage et al., in Monoclonal Antibody Production Techniques andApplications, pp. 79-97 (Marcel Dekker, Inc.: New York, 1987).

Thus, the modifier “monoclonal” indicates the character of the antibodyas being obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler and Milstein,Nature, 256:495 (1975), or may be made by recombinant DNA methods suchas described in U.S. Pat. No. 4,816,567. The “monoclonal antibodies” mayalso be isolated from phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990), for example.

“Humanized” forms of non-human (e.g. murine) antibodies are specificchimeric immunoglobulins, immunoglobulin chains, or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat, or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, the humanized antibody may comprise residues which arefound neither in the recipient antibody nor in the imported CDR orframework sequences. These modifications are made to further refine andoptimize antibody performance. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region or domain (Fc), typically that of ahuman immunoglobulin.

“Biologically active” and “desired biological activity” for the purposesherein mean having the ability to modulate apoptosis (either in anagonistic manner by inducing or stimulating apoptosis, or in anantagonistic manner by reducing or inhibiting apoptosis) in at least onetype of mammalian cell in vivo or ex vivo. The terms “apoptosis” and“apoptotic activity” are used in a broad sense and refer to the orderlyor controlled form of cell death in mammals that is typicallyaccompanied by one or more characteristic cell changes, includingcondensation of cytoplasm, loss of plasma membrane microvilli,segmentation of the nucleus, degradation of chromosomal DNA or loss ofmitochondrial function. This activity can be determined and measured,for instance, by cell viability assays, FACS analysis or DNAelectrophoresis, all of which are known in the art.

The terms “treating,” “treatment,” and “therapy” as used herein refer tocurative therapy, prophylactic therapy, and preventative therapy.

The term “mammal” as used herein refers to any mammal classified as amammal, including humans, cows, horses, dogs and cats. In a preferredembodiment of the invention, the mammal is a human.

II. Compositions and Methods of the Invention

Applicants have identified and isolated various polypeptides involved inmammalian cell apoptosis. In particular, Applicants have identified andisolated various Apo-3 polypeptides and forms thereof, referred toherein as Apo-2LI. The properties and characteristics of some of theseApo-3 and Apo-2LI polypeptides, particularly human Apo-3 and humanApo-2LI, are described in further detail in the Examples below. Basedupon the properties and characteristics of the Apo-3 and Apo-2LIpolypeptides disclosed herein, it is Applicants' present belief thatApo-3 is a member of the TNFR family.

As discussed in Example 4 below, a native sequence human Apo-3polypeptide was identified. The predicted polypeptide precursor is 417amino acids long (see FIG. 4). Hydropathy analysis (not shown) suggestedthe presence of a signal sequence (residues 1-24), followed by anextracellular domain (residues 25-198), a transmembrane domain (residues199-224), and an intracellular domain (residues 225-417) (FIG. 4; SEQ IDNO:6). The schematic diagram shown in FIG. 7 illustrates such domains,as well as the cysteine-rich domains.

As discussed in Example 1, Applicants also identified and isolated apolypeptide referred to herein as “Apo-2LI.” The predicted amino acidsequence of human Apo-2LI contains 181 amino acids (as shown in FIG. 1).It is presently believed that Apo-2LI comprising amino acid residues 1to 181 of FIG. 1 may be a soluble, truncated or secreted form of Apo-3.The human Apo-2LI having amino acids 1 to 181 of FIG. 1 is substantiallyhomologous (i.e., having at least 80% identity) to the extracellularsequence of native sequence human Apo-3 (amino acid residues 1 to 198,as shown in FIG. 4), and it is presently believed that such Apo-2LI (atleast in monomeric form) is a functional equivalent to the Apo-3.

A description follows as to how the polypeptides of the invention, aswell as chimeric molecules and antibodies, may be prepared. It iscontemplated that the methods and materials described below (and in theExamples herein) may be employed to prepare Apo-2LI, Apo-2LI chimericmolecules and anti-Apo-2LI antibodies, as well as Apo-3 polypeptides,Apo-3 chimeric molecules and anti-Apo-3 antibodies.

A. Preparation of Polypeptides and Nucleic Acids

The description below relates primarily to production of thepolypeptides by culturing cells transformed or transfected with a vectorcontaining Apo-3 or Apo-2LI nucleic acid. It is of course, contemplatedthat alternative methods, which are well known in the art, may beemployed to prepare the polypeptides.

1. Isolation of Encoding DNA

The DNA encoding the polypeptides of the invention may be obtained fromany cDNA library prepared from tissue believed to possess thepolypeptide mRNA and to express it at a detectable level. Accordingly,human Apo-2LI DNA can be conveniently obtained from a cDNA libraryprepared from human tissues, such as the bacteriophage library of humanthymus cDNA described in Example 1. The Apo-2LI-encoding gene may alsobe obtained from a genomic library or by oligonucleotide synthesis.Human Apo-3 DNA can be conveniently obtained from a cDNA libraryprepared from human tissues, such as the bacteriophage libraries ofhuman fetal heart and lung cDNA described in Example 4. TheApo-3-encoding gene may also be obtained from a genomic library or byoligonucleotide synthesis.

Libraries can be screened with probes (such as antibodies oroligonucleotides of at least about 20-80 bases) designed to identify thegene of interest or the protein encoded by it. Examples ofoligonucleotide probes are provided in Examples 1 and 4. Screening thecDNA or genomic library with the selected probe may be conducted usingstandard procedures, such as described in Sambrook et al., MolecularCloning: A Laboratory Manual (New York: Cold Spring Harbor LaboratoryPress, 1989). An alternative means to isolate the gene encoding, forinstance, Apo-3 or Apo-2LI is to use PCR methodology [Sambrook et al.,supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold SpringHarbor Laboratory Press, 1995)].

A preferred method of screening employs selected oligonucleotidesequences to screen cDNA libraries from various human tissues. Examples1 and 4 below describe techniques for screening cDNA libraries withdifferent oligonucleotide probes. The oligonucleotide sequences selectedas probes should be of sufficient length and sufficiently unambiguousthat false positives are minimized. The oligonucleotide is preferablylabeled such that it can be detected upon hybridization to DNA in thelibrary being screened. Methods of labeling are well known in the art,and include the use of radiolabels like ³²P-labeled ATP, biotinylationor enzyme labeling.

Nucleic acid having all the protein coding sequence may be obtained byscreening selected cDNA or genomic libraries using the deduced aminoacid sequence disclosed herein for the first time, and, if necessary,using conventional primer extension procedures as described in Sambrooket al., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

Apo-3 or Apo-2LI variants can be prepared by introducing appropriatenucleotide changes into the DNA of such polypeptides, or by synthesis ofthe desired polypeptide. Any combination of insertion, substitution,and/or deletion can be made to arrive at the final construct, providedthat the final construct possesses the desired activity as definedherein. Those skilled in the art will appreciate that amino acid changesmay alter post-translational processes of the Apo-3, such as changingthe number or position of glycosylation sites or altering the membraneanchoring characteristics.

Variations in the native sequence Apo-3 or Apo-2LI as described abovecan be made using any of the techniques and guidelines for conservativeand non-conservative mutations set forth in U.S. Pat. No. 5,364,934.These include oligonucleotide-mediated (site-directed) mutagenesis,alanine scanning, and PCR mutagenesis.

2. Insertion of Nucleic Acid into A Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding Apo-3 or Apo-2LImay be inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. Various vectors arepublicly available. The vector components generally include, but are notlimited to, one or more of the following: a signal sequence, an originof replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence, each of which isdescribed below.

(i) Signal Secuence Component

The polypeptides of the invention may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which may be a signal sequence or other polypeptide havinga specific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the DNA that is inserted into the vector.The heterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. The signal sequence may be a prokaryotic signal sequenceselected, for example, from the group of the alkaline phosphatase,penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeastsecretion the signal sequence may be, e.g., the yeast invertase leader,alpha factor leader (including Saccharomyces and Kluyveromyces α-factorleaders, the latter described in U.S. Pat. No. 5,010,182), or acidphosphatase leader, the C. albicans glucoamylase leader (EP 362,179published 4 Apr. 1990), or the signal described in WO 90/13646 published15 Nov. 1990. In mammalian cell expression the native Apo-3 or Apo-2LIpresequence that normally directs insertion of the polypeptide in thecell membrane of human cells in vivo is satisfactory, although othermammalian signal sequences may be used to direct secretion of theprotein, such as signal sequences from secreted polypeptides of the sameor related species, as well as viral secretory leaders, for example, theherpes simplex glycoprotein D signal.

The DNA for such precursor region is preferably ligated in reading frameto DNA encoding the polypeptide.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used because it contains the earlypromoter).

Most expression vectors are “shuttle” vectors, i.e., they are capable ofreplication in at least one class of organisms but can be transfectedinto another organism for expression. For example, a vector is cloned inE. coli and then the same vector is transfected into yeast or mammaliancells for expression even though it is not capable of replicatingindependently of the host cell chromosome.

DNA may also be amplified by insertion into the host genome. This isreadily accomplished using Bacillus species as hosts, for example, byincluding in the vector a DNA sequence that is complementary to asequence found in Bacillus genomic DNA. Transfection of Bacillus withthis vector results in homologous recombination with the genome andinsertion of the polypeptide's DNA. However, the recovery of genomic DNAencoding Apo-3 or Apo-2LI is more complex than that of an exogenouslyreplicated vector because restriction enzyme digestion is required toexcise the DNA.

(iii) Selection Gene Component

Expression and cloning vectors typically contain a selection gene, alsotermed a selectable marker. This gene encodes a protein necessary forthe survival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin [Southern et al., J. Molec. Appl. Genet., 1:327(1982)], mycophenolic acid (Mulligan et al., Science, 209:1422 (1980)]or hygromycin [Sugden et al., Mol. Cell. Biol., 5:410-413 (1985)]. Thethree examples given above employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug G418 or neomycin(geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theApo-3 or Apo-2LI nucleic acid, such as DHFR or thymidine kinase. Themammalian cell transformants are placed under selection pressure thatonly the transformants are uniquely adapted to survive by virtue ofhaving taken up the marker. Selection pressure is imposed by culturingthe transformants under conditions in which the concentration ofselection agent in the medium is successively changed, thereby leadingto amplification of both the selection gene and the DNA that encodes thepolypeptide. Amplification is the process by which genes in greaterdemand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Increased quantities of Apo-3 or Apo-2LI aresynthesized from the amplified DNA. Other examples of amplifiable genesinclude metallothionein-I and -II, adenosine deaminase, and ornithinedecarboxylase.

Cells transformed with the DHFR selection gene may first be identifiedby culturing all of the transformants in a culture medium that containsmethotrexate (Mtx), a competitive antagonist of DHFR. An appropriatehost cell when wild-type DHFR is employed is the Chinese hamster ovary(CHO) cell line deficient in DHFR activity, prepared and propagated asdescribed by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980).The transformed cells are then exposed to increased levels ofmethotrexate. This leads to the synthesis of multiple copies of the DHFRgene, and, concomitantly, multiple copies of other DNA comprising theexpression vectors, such as the DNA encoding the Apo-3 or Apo-2LI. Thisamplification technique can be used with any otherwise suitable host,e.g., ATCC No. CCL61 CHO-K1, notwithstanding the presence of endogenousDHFR if, for example, a mutant DHFR gene that is highly resistant to Mtxis employed (EP 117,060).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding Apo-3 or Apo-2LI, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979);Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157(1980)]. The trp1 gene provides a selection marker for a mutant strainof yeast lacking the ability to grow in tryptophan, for example, ATCCNo. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)]. The presence of thetrp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKDl canbe used for transformation of Kluyveromyces yeasts [Bianchi et al.,Curr. Genet., 12:185 (1987)]. More recently, an expression system forlarge-scale production of recombinant calf chymosin was reported for K.lactis [Van den Berg, Bio/Technology, 8:135 (1990)]. Stable multi-copyexpression vectors for secretion of mature recombinant human serumalbumin by industrial strains of Kluyveromyces have also been disclosed[Fleer et al., Bio/Technology, 9:968-975 (1991)].

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the Apo-3 orApo-2LI nucleic acid sequence. Promoters are untranslated sequenceslocated upstream (5′) to the start codon of a structural gene (generallywithin about 100 to 1000 bp) that control the transcription andtranslation of particular nucleic acid sequence, such as the Apo-3 orApo-2LI nucleic acid sequence, to which they are operably linked. Suchpromoters typically fall into two classes, inducible and constitutive.Inducible promoters are promoters that initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, e.g., the presence or absence of a nutrient or achange in temperature. At this time a large number of promotersrecognized by a variety of potential host cells are well known. Thesepromoters are operably linked to Apo-3 or Apo-2LI encoding DNA byremoving the promoter from the source DNA by restriction enzymedigestion and inserting the isolated promoter sequence into the vector.Both the native Apo-3 or Apo-2LI promoter sequence and many heterologouspromoters may be used to direct amplification and/or expression of DNAencoding the polypeptide.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems [Chang et al., Nature,275:617-624 (1978); Goeddel et al., Nature, 281:544 (1979)], alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tacpromoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)].However, other known bacterial promoters are suitable. Their nucleotidesequences have been published, thereby enabling a skilled workeroperably to ligate them to DNA encoding Apo-3 or Apo-2LI [Siebenlist etal., Cell, 20:269 (1980)] using linkers or adaptors to supply anyrequired restriction sites. Promoters for use in bacterial systems alsowill contain a Shine-Dalgarno (S.D.) sequence operably linked to theDNA.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:12073-12080 (1980)] or other glycolytic enzymes [Hesset al., J. Adv. Enzyme Req., 7:149 (1968); Holland, Biochemistry,17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Transcription from vectors in mammalian host cells is controlled, forexample, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and mostpreferably Simian Virus 40 (SV40), from heterologous mammalianpromoters, e.g., the actin promoter or an immunoglobulin promoter, fromheat-shock promoters, and from the promoter normally associated with theApo-3 or Apo-2LI sequence, provided such promoters are compatible withthe host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication [Fiers et al., Nature, 273:113 (1978); Mulligan and Berg,Science, 209:1422-1427 (1980); Paviakis et al., Proc. Natl. Acad. Sci.USA, 78:7398-7402 (1981)]. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment [Greenaway et al., Gene, 18:355-360 (1982)]. A system forexpressing DNA in mammalian hosts using the bovine papilloma virus as avector is disclosed in U.S. Pat. No. 4,419,446. A modification of thissystem is described in U.S. Pat. No. 4,601,978 [See also Gray et al.,Nature, 295:503-508 (1982) on expressing cDNA encoding immune interferonin monkey cells; Reyes et al., Nature, 297:598-601 (1982) on expressionof human β-interferon cDNA in mouse cells under the control of athymidine kinase promoter from herpes simplex virus; Canaani and Berg,Proc. Natl. Acad. Sci. USA 79:5166-5170 (1982) on expression of thehuman interferon β1 gene in cultured mouse and rabbit cells; and Gormanet al., Proc. Natl. Acad. Sci. USA, 79:6777-6781 (1982) on expression ofbacterial CAT sequences in CV-1 monkey kidney cells, chicken embryofibroblasts, Chinese hamster ovary cells, HeLa cells, and mouse NIH-3T3cells using the Rous sarcoma virus long terminal repeat as a promoter].

(v) Enhancer Element Component

Transcription of a DNA encoding the Apo-3 or Apo-2LI of this inventionby higher eukaryotes may be increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp, that act on a promoter to increase its transcription.Enhancers are relatively orientation and position independent, havingbeen found 5′ [Laimins et al., Proc. Natl. Acad. Sci. USA, 78:464-468(1981)] and 31 [Lusky et al., Mol. Cell Bio., 3:1108 (1983]) to thetranscription unit, within an intron [Banerji et al., Cell, 33:729(1983)], as well as within the coding sequence itself [Osborne et al.,Mol. Cell Bio., 4:1293 (1984)]. Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein, andinsulin) Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature, 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thecoding sequence, but is preferably located at a site 5′ from thepromoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding Apo-3 or Apo-2LI.

(vii) Construction and Analysis of Vectors

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and re-ligated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures can be used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res., 9:309 (1981) or by the method of Maxam et al., Methods inEnzymology, 65:499 (1980).

(viii) Transient Expression Vectors

Expression vectors that provide for the transient expression inmammalian cells of DNA encoding Apo-3 or Apo-2LI may be employed. Ingeneral, transient expression involves the use of an expression vectorthat is able to replicate efficiently in a host cell, such that the hostcell accumulates many copies of the expression vector and, in turn,synthesizes high levels of a desired polypeptide encoded by theexpression vector [Sambrook et al., supra]. Transient expressionsystems, comprising a suitable expression vector and a host cell, allowfor the convenient positive identification of polypeptides encoded bycloned DNAs, as well as for the rapid screening of such polypeptides fordesired biological or physiological properties. Thus, transientexpression systems are particularly useful in the invention for purposesof identifying Apo-3 variants or Apo-2LI variants.

(ix) Suitable Exemplary Vertebrate Cell Vectors

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of Apo-3 or Apo-2LI in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293:620-625 (1981); Mantei et al.,Nature, 281:40-46 (1979); EP 117,060; and EP 117,058. A particularlyuseful plasmid for mammalian cell culture expression of Apo-2LI is pRK7[EP 278,776; also described in Example 1].

3. Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include but are not limitedto eubacteria, such as Gram-negative or Gram-positive organisms, forexample, Enterobacteriaceae such as Escherichia, e.g., E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonellatyphimurium, Serratia, e.g., Serratia marcescans, and Shigella, as wellas Bacilli such as B. subtilis and B. licheniformis (e.g., B.licheniformis 41P disclosed in DD 266,710 published 12 Apr. 1989),Pseudomonas such as P. aeruginosa, and Streptomyces. Preferably, thehost cell should secrete minimal amounts of proteolytic enzymes.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts. Saccharomycescerevisiae, or common baker's yeast, is the most commonly used amonglower eukaryotic host microorganisms. However, a number of other genera,species, and strains are commonly available and useful herein.

Suitable host cells for the expression of glycosylated polypeptides arederived from multicellular organisms. Such host cells are capable ofcomplex processing and glycosylation activities. In principle, anyhigher eukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori havebeen identified [See, e.g., Luckow et al., Bio/Technology, 6:47-55(1988); Miller et al., in Genetic Engineering, Setlow et al., eds., Vol.8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al., Nature,315:592-594 (1985)]. A variety of viral strains for transfection arepublicly available, e.g., the L-1 variant of Autographa californica NPVand the Bm-5 strain of Bombyx mori NPV.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens. During incubation of the plant cell culturewith A. tumefaciens, the DNA encoding the polypeptide can be transferredto the plant cell host such that it is transfected, and will, underappropriate conditions, express the DNA encoding Apo-3 or Apo-2LI. Inaddition, regulatory and signal sequences compatible with plant cellsare available, such as the nopaline synthase promoter andpolyadenylation signal sequences [Depicker et al., J. Mol. Appl. Gen.,1:561 (1982)]. In addition, DNA segments isolated from the upstreamregion of the T-DNA 780 gene are capable of activating or increasingtranscription levels of plant-expressible genes in recombinantDNA-containing plant tissue [EP 321,196 published 21 Jun. 1989].

Propagation of vertebrate cells in culture (tissue culture) is also wellknown in the art [See, e.g., Tissue Culture, Academic Press, Kruse andPatterson, editors (1973)]. Examples of useful mammalian host cell linesare monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);human embryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci.,383:44-68 (1982)); MRC 5 cells; and FS4 cells.

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in Sambrook et al., supra, orelectroporation is generally used for prokaryotes or other cells thatcontain substantial cell-wall barriers. Infection with Agrobacteriumtumefaciens is used for transformation of certain plant cells, asdescribed by Shaw et al., Gene, 23:315 (1983) and WO 89/05859 published29 Jun. 1989. In addition, plants may be transfected using ultrasoundtreatment as described in WO 91/00358 published 10 Jan. 1991.

For mammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1973) is preferred. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

4. Culturing the Host Cells

Prokaryotic cells used to produce Apo-3 or Apo-2LI may be cultured insuitable media as described generally in Sambrook et al., supra.

The mammalian host cells may be cultured in a variety of media. Examplesof commercially available media include Ham's Flo (Sigma), MinimalEssential Medium (“MEM”, Sigma), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium (“DMEM”, Sigma). Any such media may besupplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleosides (such as adenosine and thymidine), antibiotics (suchas Gentamycin™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

In general, principles, protocols, and practical techniques formaximizing the productivity of mammalian cell cultures can be found inMammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRLPress, 1991).

The host cells referred to in this disclosure encompass cells in cultureas well as cells that are within a host animal.

5. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, and particularly ³²P. However, other techniques may alsobe employed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionucleotides, fluorescers or enzymes. Alternatively,antibodies may be employed that can recognize specific duplexes,including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes orDNA-protein duplexes. The antibodies in turn may be labeled and theassay may be carried out where the duplex is bound to a surface, so thatupon the formation of duplex on the surface, the presence of antibodybound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. With immunohistochemicalstaining techniques, a cell sample is prepared, typically by dehydrationand fixation, followed by reaction with labeled antibodies specific forthe gene product coupled, where the labels are usually visuallydetectable, such as enzymatic labels, fluorescent labels, or luminescentlabels.

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared in any mammal. Conveniently, the antibodies may be preparedagainst a native sequence Apo-3 polypeptide or Apo-2LI polypeptide oragainst a synthetic peptide based on the DNA sequences provided hereinor against exogenous sequence fused to Apo-3 or Apo-2LI DNA and encodinga specific antibody epitope.

6. Purification of Polypeptide

Apo-2LI preferably is recovered from the culture medium as a secretedpolypeptide, although it also may be recovered from host cell lysateswhen directly produced without a secretory signal. Forms of Apo-3 may berecovered from culture medium or from host cell lysates. If the Apo-3 orApo-2LI is membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g. Triton-X 100) or its extracellularregion may be released by enzymatic cleavage.

When Apo-3 or Apo-2LI is produced in a recombinant cell other than oneof human origin, the polypeptide is free of proteins or polypeptides ofhuman origin. However, it may be desired to purify the polypeptide fromrecombinant cell proteins or polypeptides to obtain preparations thatare substantially homogeneous as to Apo-3 or Apo-2LI. As a first step,the culture medium or lysate may be centrifuged to remove particulatecell debris. Apo-3 or Apo-2LI thereafter is purified from contaminantsoluble proteins and polypeptides, with the following procedures beingexemplary of suitable purification procedures: by fractionation on anion-exchange column; ethanol precipitation; reverse phase HPLC;chromatography on silica or on a cation-exchange resin such as DEAE;chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; and protein A Sepharosecolumns to remove contaminants such as IgG.

Apo-3 or Apo-2LI variants in which residues have been deleted, inserted,or substituted can be recovered in the same fashion as native sequencepolypeptides, taking account of any substantial changes in propertiesoccasioned by the variation. For example, preparation of an Apo-3 fusionwith another protein or polypeptide, e.g., a bacterial or viral antigen,immunoglobulin sequence, or receptor sequence, may facilitatepurification; an immunoaffinity column containing antibody to thesequence can be used to adsorb the fusion polypeptide. Other types ofaffinity matrices also can be used.

A protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) alsomay be useful to inhibit proteolytic degradation during purification,and antibiotics may be included to prevent the growth of adventitiouscontaminants. One skilled in the art will appreciate that purificationmethods suitable for the native sequence polypeptide may requiremodification to account for changes in the character of the polypeptideor its variants upon expression in recombinant cell culture.

7. Covalent Modifications of Polypeptides

Covalent modifications of Apo-3 or Apo-2LI are included within the scopeof this invention. One type of covalent modification of thesepolypeptides is introduced into the molecule by reacting targeted aminoacid residues with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues.

Derivatization with bifunctional agents is useful for crosslinking thepolypeptide to a water-insoluble support matrix or surface for use inthe method for purifying anti-Apo-3 or anti-Apo-2LI antibodies, andvice-versa. Derivatization with one or more bifunctional agents willalso be useful for crosslinking, for instance, Apo-3 molecules togenerate Apo-3 dimers. Such dimers may increase binding avidity andextend half-life of the molecule in vivo. Commonly used crosslinkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)-dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group. The modifiedforms of the residues fall within the scope of the present invention.

Another type of covalent modification included within the scope of thisinvention comprises altering the native glycosylation pattern of thepolypeptide. “Altering the native glycosylation pattern” is intended forpurposes herein to mean deleting one or more carbohydrate moieties foundin native sequence Apo-3 or Apo-2LI, and/or adding one or moreglycosylation sites that are not present in the native sequence Apo-3 orApo-2LI, respectively.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxylamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites may be accomplished by altering theamino acid sequence such that it contains one or more of theabove-described tripeptide sequences (for N-linked glycosylation sites).The alteration may also be made by the addition of, or substitution by,one or more serine or threonine residues to the native sequence Apo-3 orApo-2LI (for O-linked glycosylation sites). The amino acid sequence mayoptionally be altered through changes at the DNA level, particularly bymutating the DNA encoding the Apo-3 or Apo-2LI polypeptide atpreselected bases such that codons are generated that will translateinto the desired amino acids. The DNA mutation(s) may be made usingmethods described above and in U.S. Pat. No. 5,364,934, supra.

Another means of increasing the number of carbohydrate moieties on thepolypeptide is by chemical or enzymatic coupling of glycosides to thepolypeptide. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330 published 11 Sep. 1987, and in Aplin andWriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. For instance, chemical deglycosylation by exposing thepolypeptide to the compound trifluoromethanesulfonic acid, or anequivalent compound can result in the cleavage of most or all sugarsexcept the linking sugar (N-acetylglucosamine or N-acetylgalactosamine),while leaving the polypeptide intact. Chemical deglycosylation isdescribed by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987)and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavageof carbohydrate moieties on polypeptides can be achieved by the use of avariety of endo- and exo-glycosidases as described by Thotakura et al.,Meth. Enzymol., 138:350 (1987).

Glycosylation at potential glycosylation sites may be prevented by theuse of the compound tunicamycin as described by Duksin et al., J. Biol.Chem., 257:3105-3109 (1982). Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification comprises linking the Apo-3 orApo-2LI polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

8. Chimeric Molecules

The present invention also provides chimeric molecules comprising Apo-3or Apo-2LI fused to another, heterologous polypeptide or amino acidsequence.

In one embodiment, the chimeric molecule comprises a fusion of the Apo-3or Apo-2LI with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the Apo-3 or Apo-2LI,respectively. The presence of such epitope-tagged forms can be detectedusing an antibody against the tag polypeptide. Also, provision of theepitope tag enables the Apo-3 or Apo-2LI to be readily purified byaffinity purification using an anti-tag antibody or another type ofaffinity matrix that binds to the epitope tag.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include the flu HA tag polypeptide and its antibody12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myctag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan etal., Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the HerpesSimplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al.,Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptidesinclude the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210(1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194(1992)]; an α-tubulin epitope peptide [Skinner et al., J. Biol. Chem.,266:14163-14166 (1991)]; and the T7 gene 10 protein peptide tag[Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397(1990)]. Once the tag polypeptide has been selected, an antibody theretocan be generated using the techniques disclosed herein.

Generally, epitope-tagged-Apo-3 or Apo-2LI may be constructed andproduced according to the methods described above. Apo-3 or Apo-2LI-tagpolypeptide fusions are preferably constructed by fusing the cDNAsequence encoding the Apo-3 or Apo-2LI portion in-frame to the tagpolypeptide DNA sequence and expressing the resultant DNA fusionconstruct in appropriate host cells. Ordinarily, when preparing the tagpolypeptide chimeras of the present invention, nucleic acid encoding theApo-3 or Apo-2LI will be fused at its 3′ end to nucleic acid encodingthe N-terminus of the tag polypeptide, however 5′ fusions are alsopossible. For example, a polyhistidine sequence of about 5 to about 10histidine residues may be fused at the N-terminus or the C-terminus andused as a purification handle in affinity chromatography.

Epitope-tagged Apo-3 or Apo-2LI can be purified by affinitychromatography using the anti-tag antibody. The matrix to which theaffinity antibody is attached may include, for instance, agarose,controlled pore glass or poly(styrenedivinyl)benzene. The epitope-taggedpolypeptide can then be eluted from the affinity column using techniquesknown in the art.

In another embodiment, the chimeric molecule comprises an Apo-3 orApo-2LI polypeptide fused to an immunoglobulin sequence or otherheterologous sequence. Optionally, the chimeric molecule comprises anApo-2LI fused to an immunoglobulin constant domain or TNFR sequence. Thechimeric molecule may also comprise a particular domain sequence ofApo-3, such as the extracellular domain sequence of native Apo-3 fusedto an immunoglobulin sequence. This includes chimeras in monomeric,homo- or heteromultimeric, and particularly homo- or heterodimeric, or-tetrameric forms; optionally, the chimeras may be in dimeric forms orhomodimeric heavy chain forms. Generally, these assembledimmunoglobulins will have known unit structures as represented by thefollowing diagrams.

A basic four chain structural unit is the form in which IgG, IgD, andIgE exist. A four chain unit is repeated in the higher molecular weightimmunoglobulins; IgM generally exists as a pentamer of basic four-chainunits held together by disulfide bonds. IgA globulin, and occasionallyIgG globulin, may also exist in a multimeric form in serum. In the caseof multimers, each four chain unit may be the same or different.

The following diagrams depict some exemplary monomer, homo- andheterodimer and homo- and heteromultimer structures. These diagrams aremerely illustrative, and the chains of the multimers are believed to bedisulfide bonded in the same fashion as native immunoglobulins.

In the foregoing diagrams, “A” means an Apo-3 sequence, Apo-2LIsequence, or an Apo-3 or Apo-2LI sequence fused to a heterologoussequence; X is an additional agent, which may be the same as A ordifferent, a portion of an immunoglobulin superfamily member such as avariable region or a variable region-like domain, including a native orchimeric immunoglobulin variable region, a toxin such a pseudomonasexotoxin or ricin, or a sequence functionally binding to anotherprotein, such as other cytokines (i.e., IL-1, interferon-γ) or cellsurface molecules (i.e., NGFR, CD40, OX40, Fas antigen, T2 proteins ofShope and myxoma poxviruses), or a polypeptide therapeutic agent nototherwise normally associated with a constant domain; Y is a linker oranother receptor sequence; and V_(L), V_(H), C_(L) and C_(H) representlight or heavy chain variable or constant domains of an immunoglobulin.Structures comprising at least one CRD of an Apo-3 or Apo-2LI sequenceas “A” and another cell-surface protein having a repetitive pattern ofCRDs (such as TNFR) as “X” are specifically included.

It will be understood that the above diagrams are merely exemplary ofthe possible structures of the chimeras of the present invention, and donot encompass all possibilities. For example, there might desirably beseveral different “A”s, “X”s, or “Y”s in any of these constructs. Also,the heavy or light chain constant domains may be originated from thesame or different immunoglobulins. All possible permutations of theillustrated and similar structures are all within the scope of theinvention herein.

In general, the chimeric molecules can be constructed in a fashionsimilar to chimeric antibodies in which a variable domain from anantibody of one species is substituted for the variable domain ofanother species. See, for example, EP 0 125 023; EP 173,494; Munro,Nature, 312:597 (13 Dec. 1984); Neuberger et al., Nature, 312:604-608(13 Dec. 1984); Sharon et al., Nature, 309:364-367 (24 May 1984);Morrison et al., Proc. Nat'l. Acad. Sci. USA, 81:6851-6855 (1984);Morrison et al., Science, 229:1202-1207 (1985); Boulianne et al.,Nature, 312:643-646 (13 Dec. 1984); Capon et al., Nature, 337:525-531(1989); Traunecker et al., Nature, 339:68-70 (1989).

Alternatively, the chimeric molecules may be constructed as follows. TheDNA including a region encoding the desired sequence, such as an Apo-3,Apo-2LI and/or TNFR sequence, is cleaved by a restriction enzyme at orproximal to the 3′ end of the DNA encoding the immunoglobulin-likedomain(s) and at a point at or near the DNA encoding the N-terminal endof the Apo-3, Apo-2LI, or TNFR polypeptide (where use of a differentleader is contemplated) or at or proximal to the N-terminal codingregion for TNFR (where the native signal is employed). This DNA fragmentthen is readily inserted proximal to DNA encoding an immunoglobulinlight or heavy chain constant region and, if necessary, the resultingconstruct tailored by deletional mutagenesis. Preferably, the Ig is ahuman immunoglobulin when the chimeric molecule is intended for in vivotherapy for humans. DNA encoding immunoglobulin light or heavy chainconstant regions is known or readily available from cDNA libraries or issynthesized. See for example, Adams et al., Biochemistry, 19:2711-2719(1980); Gough et al., Biochemistry, 19:2702-2710 (1980); Dolby et al.,Proc. Natl. Acad. Sci. USA, 77:6027-6031 (1980); Rice et al., Proc.Natl. Acad. Sci., 79:7862-7865 (1982); Falkner et al., Nature,298:286-288 (1982); and Morrison et al., Ann. Rev. Immunol., 2:239-256(1984).

Further details of how to prepare such fusions are found in publicationsconcerning the preparation of immunoadhesins. Immunoadhesins in general,and CD4-Ig fusion molecules specifically are disclosed in WO 89/02922,published 6 Apr. 1989). Molecules comprising the extracellular portionof CD4, the receptor for human immunodeficiency virus (HIV), linked toIgG heavy chain constant region are known in the art and have been foundto have a markedly longer half-life and lower clearance than the solubleextracellular portion of CD4 [Capon et al., supra; Byrn et al., Nature,344:667 (1990)]. The construction of specific chimeric TNFR-IgGmolecules is also described in Ashkenazi et al. Proc. Natl. Acad. Sci.,88:10535-10539 (1991); Lesslauer et al. [J. Cell. Biochem. Supplement15F, 1991, p. 115 (P 432)]; and Peppel and Beutler, J. Cell. Biochem.Supplement 15F, 1991, p. 118 (P 439)]. B. Therapeutic andNon-therapeutic Uses for Apo-3 and Apo-2LI

Apo-3, as disclosed in the present specification, can be employedtherapeutically to induce apoptosis or NF-κB or JNK mediated geneexpression in mammalian cells. This therapy can be accomplished forinstance, using in vivo or ex vivo gene therapy techniques and includesthe use of the death domain sequences disclosed herein. The Apo-3chimeric molecules (including the chimeric molecules containing theextracellular domain sequence of Apo-3) comprising immunoglobulinsequences can also be employed therapeutically to inhibit apoptosis orNF-κB induction or JNK activation. Apo-2LI, as disclosed in theapplication, can be employed therapeutically to inhibit mammalian cellapoptosis in vivo or ex vivo. Generally, the methods comprise exposingthe cells to an effective amount of the Apo-2LI.

The Apo-3 and Apo-2LI of the invention also have utility innon-therapeutic applications. Nucleic acid sequences encoding the Apo-3or Apo-2LI may be used as a diagnostic for tissue-specific typing. Forexample, procedures like in situ hybridization, Northern and Southernblotting, and PCR analysis may be used to determine whether DNA and/orRNA encoding the polypeptide is present in the cell type(s) beingevaluated. Apo-3 or Apo-2LI nucleic acid will also be useful for thepreparation of Apo-3 or Apo-2LI, respectively by the recombinanttechniques described herein.

The isolated Apo-3 or Apo-2LI may be used in quantitative diagnosticassays as a control against which samples containing unknown quantitiesof Apo-3 or Apo-2LI may be prepared. Apo-3 preparations are also usefulin generating antibodies, as standards in assays for Apo-3 or Apo-2LI(e.g., by labeling Apo-3 or Apo-2LI for use as a standard in aradioimmunoassay, radioreceptor assay, or enzyme-linked immunoassay), inaffinity purification techniques, and in competitive-type receptorbinding assays when labeled with, for instance, radioiodine, enzymes, orfluorophores.

Modified forms of the Apo-3, such as the Apo-3-IgG chimeric molecules(immunoadhesins) described above, can be used as immunogens in producinganti-Apo-3 antibodies.

Nucleic acids which encode Apo-3 or its modified forms can also be usedto generate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA which is integrated into thegenome of a cell from which a transgenic animal develops. In oneembodiment, cDNA encoding Apo-3 or an appropriate sequence thereof canbe used to clone genomic DNA encoding Apo-3 in accordance withestablished techniques and the genomic sequences used to generatetransgenic animals that contain cells which express DNA encoding Apo-3.Methods for generating transgenic animals, particularly animals such asmice or rats, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009. Typically,particular cells would be targeted for Apo-3 transgene incorporationwith tissue-specific enhancers. Transgenic animals that include a copyof a transgene encoding Apo-3 introduced into the germ line of theanimal at an embryonic stage can be used to examine the effect ofincreased expression of DNA encoding Apo-3. Such animals can be used astester animals for reagents thought to confer protection from, forexample, pathological conditions associated with excessive apoptosis. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition. Inanother embodiment, transgenic animals that carry a soluble form ofApo-3 such as the Apo-3 ECD or an immunoglobulin chimera of such formcould be constructed to test the effect of chronic neutralization of theligand of Apo-3.

Alternatively, non-human homologues of Apo-3 can be used to construct aApo-3 “knock out” animal which has a defective or altered gene encodingApo-3 as a result of homologous recombination between the endogenousgene encoding Apo-3 and altered genomic DNA encoding Apo-3 introducedinto an embryonic cell of the animal. For example, cDNA encoding Apo-3can be used to clone genomic DNA encoding Apo-3 in accordance withestablished techniques. A portion of the genomic DNA encoding Apo-3 canbe deleted or replaced with another gene, such as a gene encoding aselectable marker which can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends)are included in the vector [see e.g., Thomas and Capecchi, Cell, 51:503(1987) for a description of homologous recombination vectors]. Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected [see e.g., Li et al.,Cell, 69:915 (1992)]. The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987),pp. 113-151]. A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the Apo-3 polypeptide, including forexample, development of tumors.

C. Antibody Preparation

The present invention further provides anti-Apo-3 antibodies andanti-Apo-2LI antibodies. Such antibodies may be prepared as follows.Exemplary antibodies include polyclonal, monoclonal, humanized,bispecific, and heteroconjugate antibodies.

1. Polyclonal Antibodies

The antibodies may comprise polyclonal antibodies. Methods of preparingpolyclonal antibodies are known to the skilled artisan. Polyclonalantibodies can be raised in a mammal, for example, by one or moreinjections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the Apo-3 or Apo-2LI polypeptide or afusion protein thereof. An example of a suitable immunizing agent is aApo-3-IgG fusion protein or chimeric molecule (including an Apo-3ECD-IgG fusion protein). Cells expressing Apo-3 at their surface mayalso be employed. It may be useful to conjugate the immunizing agent toa protein known to be immunogenic in the mammal being immunized.Examples of such immunogenic proteins which may be employed include butare not limited to keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor. An aggregating agent suchas alum may also be employed to enhance the mammal's immune response.Examples of adjuvants which may be employed include Freund's completeadjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetictrehalose dicorynomycolate). The immunization protocol may be selectedby one skilled in the art without undue experimentation. The mammal canthen be bled, and the serum assayed for antibody titer. If desired, themammal can be boosted until the antibody titer increases or plateaus.

2. Monoclonal Antibodies

The antibodies may, alternatively, be monoclonal antibodies. Monoclonalantibodies may be prepared using hybridoma methods, such as thosedescribed by Kohler and Milstein, supra. In a hybridoma method, a mouse,hamster, or other appropriate host animal, is typically immunized (suchas described above) with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

The immunizing agent will typically include the Apo-3 or Apo-2LIpolypeptide or a fusion protein thereof. An example of a suitableimmunizing agent is a Apo-3-IgG fusion protein or chimeric molecule.Cells expressing Apo-3 or Apo-2LI at their surface may also be employed.Generally, either peripheral blood lymphocytes (“PBLs”) are used ifcells of human origin are desired, or spleen cells or lymph node cellsare used if non-human mammalian sources are desired. The lymphocytes arethen fused with an immortalized cell line using a suitable fusing agent,such as polyethylene glycol, to form a hybridoma cell [Goding,Monoclonal Antibodies: Principles and Practice, Academic Press, (1986)pp. 59-103]. Immortalized cell lines are usually transformed mammaliancells, particularly myeloma cells of rodent, bovine and human origin.Usually, rat or mouse myeloma cell lines are employed. The hybridomacells may be cultured in a suitable culture medium that preferablycontains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells. For example, if the parental cells lackthe enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT orHPRT), the culture medium for the hybridomas typically will includehypoxanthine, aminopterin, and thymidine (“HAT medium”), whichsubstances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Rockville, Md. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against Apo-3or Apo-2LI. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography. The monoclonal antibodies may alsobe made by recombinant DNA methods, such as those described in U.S. Pat.No. 4,816,567. DNA encoding the monoclonal antibodies of the inventioncan be readily isolated and sequenced using conventional procedures(e.g., by using oligonucleotide probes that are capable of bindingspecifically to genes encoding the heavy and light chains of murineantibodies). The hybridoma cells of the invention serve as a preferredsource of such DNA. Once isolated, the DNA may be placed into expressionvectors, which are then transfected into host cells such as simian COScells, Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. The DNA also may bemodified, for example, by substituting the coding sequence for humanheavy and light chain constant domains in place of the homologous murinesequences [U.S. Pat. No. 4,816,567; Morrison et al., supra] or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the invention, or can be substituted for thevariable domains of one antigen-combining site of an antibody of theinvention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 published Dec. 22, 1994and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typicallyproduces two identical antigen binding fragments, called Fab fragments,each with a single antigen binding site, and a residual Fc fragment.Pepsin treatment yields an F(ab′)₂ fragment that has two antigencombining sites and is still capable of cross-linking antigen.

The Fab fragments produced in the antibody digestion also contain theconstant domains of the light chain and the first constant domain (CH₁)of the heavy chain. Fab′ fragments differ from Fab fragments by theaddition of a few residues at the carboxy terminus of the heavy chainCH₁ domain including one or more cysteines from the antibody hingeregion. Fab′-SH is the designation herein for Fab′ in which the cysteineresidue(s) of the constant domains bear a free thiol group. F(ab′)₂antibody fragments originally were produced as pairs of Fab′ fragmentswhich have hinge cysteines between them. Other chemical couplings ofantibody fragments are also known.

3. Humanized Antibodies

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-327 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important in order to reduceantigenicity. According to the “best-fit” method, the sequence of thevariable domain of a rodent antibody is screened against the entirelibrary of known human variable domain sequences. The human sequencewhich is closest to that of the rodent is then accepted as the humanframework (FR) for the humanized antibody [Sims et al., J. Immunol.,151:2296 (1993); Chothia and Lesk, J. Mol. Biol., 196:901 (1987)].Another method uses a particular framework derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies [Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol., 151:2623 (1993)].

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products using threedimensional models of the parental and humanized sequences. Threedimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequence so that thedesired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding[see, WO 94/04679 published 3 Mar. 1994].

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region (JH) gene in chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production.Transfer of the human germ-line immunoglobulin gene array in suchgerm-line mutant mice will result in the production of human antibodiesupon antigen challenge [see, e.g., Jakobovits et al., Proc. Natl. Acad.Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258(1993); Bruggemann et al., Year in Immuno., 7:33 (1993)]. Humanantibodies can also be produced in phage display libraries [Hoogenboomand Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)]. The techniques of Cole et al. and Boerner et al. arealso available for the preparation of human monoclonal antibodies (Coleet al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)].

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe Apo-3 or the Apo-2LI, the other one is for any other antigen, andpreferably for a cell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy-chain constantdomain, comprising at least part of the hinge, CH2, and CH3 regions. Itis preferred to have the first heavy-chain constant region (CH1)containing the site necessary for light-chain binding present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy-chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance. In a preferred embodiment of this approach,the bispecific antibodies are composed of a hybrid immunoglobulin heavychain with a first binding specificity in one arm, and a hybridimmunoglobulin heavy-chain/light-chain pair (providing a second bindingspecificity) in the other arm. It was found that this asymmetricstructure facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations, as the presence of animmunoglobulin light chain in only one half of the bispecific moleculeprovides for a facile way of separation. This approach is disclosed inWO 94/04690 published 3 Mar. 1994. For further details of generatingbispecific antibodies see, for example, Suresh et al., Methods inEnzymology, 121:210 (1986).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/20373; EP 03089).It is contemplated that the antibodies may be prepared in vitro usingknown methods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.Pat. No. 4,676,980.

D. Antibody Therapeutic and Non-Therapeutic Uses

The antibodies of the invention have therapeutic utility. AgonisticApo-3 antibodies, for instance, may be employed to activate or stimulateapoptosis in cancer cells. Alternatively, antagonistic antibodies may beused to block excessive apoptosis (for instance in neurodegenerativedisease) or to block potential autoimmune/inflammatory effects of Apo-3resulting from NF-κB activation and/or JNK activation.

The antibodies may further be used in diagnostic assays. For example,Apo-3 antibodies or Apo-2LI antibodies may be used in diagnostic assaysfor Apo-3 or Apo-2LI, respectively, e.g., detecting its expression inspecific cells, tissues, or serum. Various diagnostic assay techniquesknown in the art may be used, such as competitive binding assays, director indirect sandwich assays and immunoprecipitation assays conducted ineither heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies:A Manual of Techniques, CRC Press, Inc. (1987) pp. 147-158]. Theantibodies used in the diagnostic assays can be labeled with adetectable moiety. The detectable moiety should be capable of producing,either directly or indirectly, a detectable signal. For example, thedetectable moiety may be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or¹²⁵I, a fluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkalinephosphatase, beta-galactosidase or horseradish peroxidase. Any methodknown in the art for conjugating the antibody to the detectable moietymay be employed, including those methods described by Hunter et al.,Nature, 144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Painet al., J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

Apo-3 or Apo-2LI antibodies also are useful for the affinitypurification of Apo-3 or Apo-2LI from recombinant cell culture ornatural sources. In this process, the antibodies are immobilized on asuitable support, such a Sephadex resin or filter paper, using methodswell known in the art. The immobilized antibody then is contacted with asample containing the Apo-3 or Apo-2LI to be purified, and thereafterthe support is washed with a suitable solvent that will removesubstantially all the material in the sample except the Apo-3 orApo-2LI, which is bound to the immobilized antibody. Finally, thesupport is washed with another suitable solvent that will release theApo-3 or Apo-2LI from the antibody.

E. Kits and Articles of Manufacture

In a further embodiment of the invention, there are provided articles ofmanufacture and kits containing Apo-3, Apo-2LI, or Apo-3 or Apo-2LIantibodies which can be used, for instance, for the therapeutic ornon-therapeutic applications described above. The article of manufacturecomprises a container with a label. Suitable containers include, forexample, bottles, vials, and test tubes. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which includes an active agent that is effective fortherapeutic or non-therapeutic applications, such as described above.The active agent in the composition is Apo-3 or Apo-2LI, or an Apo-3 orApo-2LI antibody. The label on the container indicates that thecomposition is used for a specific therapy or non-therapeuticapplication, and may also indicate directions for either in vivo or invitro use, such as those described above.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All references cited in the present specification are herebyincorporated by reference in their entirety.

EXAMPLES

All restriction enzymes referred to in the examples were purchased fromNew England Biolabs and used according to manufacturer's instructions.All other commercially available reagents referred to in the exampleswere used according to manufacturer's instructions unless otherwiseindicated. The source of those cells identified in the followingexamples, and throughout the specification, by ATCC accession numbers isthe American Type Culture Collection, Rockville, Md.

Example 1 Isolation of cDNA clones Encoding Human Apo-2LI

To isolate a cDNA for Apo-2LI, a lambda gt10 bacteriophage library ofhuman thymus cDNA (about 1×10⁶ clones) (HL1074a, commercially availablefrom Clontech) was screened by hybridization with syntheticoligonucleotide probes based on an EST sequence (GenBank locus H41522),which showed some degree of homology to human Fas/Apo-1. The ESTsequence of H41522 is 433 bp and when translated in its +1 frame, shows20 identities to a 78 amino acid region of human Fas/Apo-1. The sequenceof H41522 is as follows:

(SEQ ID NO:2) CTGCTGGGGGCCCGGGCCAGNGGCGGCACTCGTAGCCCCAGGTGTGACTGTGCCGGTGACTTCCACAAGAAGATTGGTCTGTTTTGTTGCAGAGGCTGCCCAGCGGGGCAACTACCTGAAGGCCCCTTGCACGGAGCCCTGCGCAACTCCACCTGCCTTGTGTGTCCCCAAGACACCTTCTTGGCCTGGGAGAACCACCATAATTCTGAATGTGCCCGCTGCCAGGCCTGTGATGAGCAGGCCTCCCAGGTGGCGCTGGAGAACTGTTCAGCAGTGGCCGACACCCGCTGTGGCTGTAAGCAGGGCTGGTTTGTGGAGTGCCAGGGTCAGCCAATGTGTCAGCAGTTTCACCCTTCTAATGCCAACCATGCCTAGACTGCGGGGCCCTGCAACGCAACACACGGCTAATNTGTTTCCCGCAGAGATNATTGTTThe oligonucleotide probes employed in the screening were 28 bp long,with the following respective sequences:

CCCGCTGCCAGGCCTGTGATGAGCAGGC (SEQ ID NO:3) CAGGGCCCCGCAGTCTAGGCATGGTTGG(SEQ ID NO:4)Hybridization was conducted with a 1:1 mixture of the two probesovernight at room temperature in buffer containing 20% formamide, 5×SSC,10% dextran sulfate, 0.1% NaPiPO₄₁ 0.05M NaPO₄, 0.05 mg salmon spermDNA, and 0.1% sodium dodecyl sulfate, followed consecutively by one washat room temperature in 6×SSC, two washes at 37° C. in 1×SSC/0.1% SDS,two washes at 37° C. in 0.5×SSC/0.1% SDS, and two washes at 37° C. in0.2×SSC/0.1% SDS. Four positive clones were identified in the cDNAlibrary, and the positive clones were confirmed to be specific by PCRusing the above hybridization probes as PCR primers. Single phageplaques containing each of the four positive clones were isolated bylimiting dilution and the DNA was purified using a Wizard Lambda PrepDNA purification kit (commercially available from Promega).

The cDNA inserts from the four bacteriophage clones were excised fromthe vector arms by digestion with EcoRI, gel-purified, and subclonedinto pRK7 [EP 278,776 published Aug. 17, 1988] that was predigested withEcoRI. Three of the clones (18.1, 24.1, and 28.1) contained an identicalopen reading frame; therefore further analysis was done with only oneclone, 18.1. Clone 18.1 was approximately 1.4 kb long.

The entire nucleotide sequence of Apo-2LI is shown in FIG. 1 (SEQ IDNO:5). The cDNA contained one open reading frame with a translationalinitiation site assigned to the ATG codon at nucleotide positions377-379. The surrounding sequence at this site is in reasonableagreement with the proposed consensus sequence for initiation sites[Kozak, J. Cell. Biol., 115:887-903 (1991)]. The open reading frame endsat the termination codon TAA at nucleotide positions 919-921.

The predicted amino acid sequence of the Apo-2LI encoded by clone 18.1contains 181 amino acids, and has a calculated molecular weight ofapproximately 19.3 kDa and an isoelectric point of approximately 7.1.Hydropathy analysis indicated the presence of a hydrophobic signalsequence at the N-terminus of approximately 20 amino acids. Twopotential N-linked glycosylation sites are located at residues 67 and105 of the polypeptide precursor.

An alignment (using the Align™ computer program) of the amino acidsequence encoded by clone 18.1 with the extracellular regions of otherknown members of the human TNF receptor family showed the followingpercentages of identity: 30.2% identity to Fas/Apo-1; 28.7% to type 1TNF receptor (TNFR1); 22.5% to the low affinity NGF receptor (LNGFR) andto CD40; 21.8% to CD30; 21.5% to CD27; 21.4% to OX40; 20.5% to type 2TNF receptor (TNFR2); 20.1% to TNF receptor related protein (TNFRrp).(See also, FIG. 2)

TNF receptor family proteins are typically characterized by the presenceof multiple (usually four) cysteine-rich domains in their extracellularregions—each cysteine-rich domain being approximately 45 amino acid longand contains approximately 6, regularly spaced, cysteine residues. Basedon the crystal structure of the type 1 TNF receptor, the cysteines ineach domain typically form three disulfide bonds in which usuallycysteines 1 and 2, 3 and 5, and 4 and 6 are paired together. Applicantsfound that the polypeptide encoded by clone 18.1 contains threecysteine-rich domains and an apparently truncated fourth cysteine-richdomain that contains only three cysteines and stops 5 amino acidsC-terminally to the third cysteine.

Amino acids 1 to 181 of the Apo-2LI clone 18.1 shown in FIG. 1 (SEQ IDNO:1) are identical to amino acids 1 to 181 of the Apo-3 polypeptide, asdescribed in Example 4 below, and shown in FIG. 4 (SEQ ID NO:6).Compared to Apo-3 polypeptide described in Example 4 below, thepolypeptide encoded by clone 18.1 is truncated within the C-terminalregion of the ECD and lacks some extracellular sequence as well as thetransmembrane and cytoplasmic sequences of Apo-3. The truncation isbelieved to occur by alternative splicing of the mRNA which introduces astop codon 5 amino acids downstream of the third cysteine of the fourthcysteine-rich domain. The 3′ untranslated region is distinct from thatof the Apo-3 clone FL8A.53 and contains a distinct polyadenylation site,suggesting that clone 18.1 represents a naturally-occurring mRNA.

Example 2 Expression of Apo-2LI Clone 18.1

A pRK7 plasmid (described in Example 1) containing the Apo-2LI cDNA (asdescribed in Example 1) in the forward orientation, or a control pRK5plasmid [Schall et al., Cell, 61:361-370 (1990); Suva, Science,237:893-896 (1987)] containing the Apo-2LI cDNA in the reverseorientation, were transfected transiently into human 293 cells (ATCC CRL1573) by calcium phosphate precipitation. After 24 hours, the medium wasreplaced by serum free medium, and the cells were incubated for anadditional 48 hours. The serum free conditioned media were thencollected, cleared by centrifugation, and concentrated 5-fold bycentrifugation in centricon tubes.

Example 3 Expression of Apo-2LI Immunoadhesin

An immunoadhesin was constructed that consisted of the Apo-2LI codingregion (as described in Example 1), including its endogenous signalsequence, fused C-terminally to residues 183-211 of type 1 TNF receptor,which was fused in turn to the hinge and Fc regions of human IgG1 heavychain, as described previously by Ashkenazi et al., supra.

The pRK5 plasmid encoding the chimeric Apo-2LI immunoadhesin wastransiently transfected into human 293 cells (described in Example 2) bycalcium phosphate precipitation. After 24 hours, the medium was replacedby serum free medium, and the cells were incubated for an additional 6days. The serum free conditioned media were then collected, cleared bycentrifugation, and purified by protein A affinity chromatography, asdescribed previously by Ashkenazi et al., supra. Gel electrophoresisshowed that the purified protein exhibited a molecular weight ofapproximately 110 kDa under non-reducing conditions (FIG. 3, lanes 3-5)and approximately 55 kDa under reducing conditions (100 mM DTT, FIG. 3,lanes 7-9), thus indicating a disulfide-bonded homodimeric immunoadhesinstructure. Higher molecular weight bands observed for non-reducingconditions are believed to be due to some aggregation of theimmunoadhesin during sample preparation.

Example 4 Isolation of cDNA Clones Encoding Human Apo-3

Human fetal heart and human fetal lung lgt10 bacteriophage cDNAlibraries (both purchased from Clontech) were screened by hybridizationwith synthetic oligonucleotide probes based on an EST (Genbank locusW71984), which showed some degree of homology to the intracellulardomain (ICD) of human TNFR1 and CD95. W71984 is a 523 bp EST, which inits −1 reading frame has 27 identities to a 43 amino acid long sequencein the ICD of human TNFR1. The oligonucleotide probes used in thescreening were 27 and 25 bp long, respectively, with the followingsequences:

GGCGCTCTGGTGGCCCTTGCAGAAGCC [SEQ ID NO:7] and TTCGGCCGAGAAGTTGAGAAATGTC.[SEQ ID NO:8]

Hybridization was done with a 1:1 mixture of the two probes overnight atroom temperature in buffer containing 20% formamide, 5×SSC, 10% dextransulfate, 0.1% NaPiPO₄, 0.05 M NaPO₄, 0.05 mg salmon sperm DNA, and 0.1%sodium dodecyl sulfate (SDS), followed consecutively by one wash at roomtemperature in 6×SSC, two washes at 37° C. in 1×SSC/0.1% SDS, two washesat 37° C. in 0.5×SSC/0.1% SDS, and two washes at 37° C. in 0.2×SSC/0.1%SDS. One positive clone from each of the fetal heart (FH20A.57) andfetal lung (FL8A.53) libraries were confirmed to be specific by PCRusing the respective above hybridization probes as primers. Single phageplaques containing each of the positive clones were isolated by limitingdilution and the DNA was purified using a Wizard lambda prep DNApurification kit (Promega).

The cDNA inserts were excised from the lambda vector arms by digestionwith EcoRI, gel-purified, and subcloned into pRK5 that was predigestedwith EcoRI. The clones were then sequenced in entirety.

Clone FH20A.57 (also referred to as Apo 3 clone FH20.57 deposited asATCC 55820, as indicated below) contains a single open reading framewith an apparent translational initiation site at nucleotide positions89-91 and ending at the stop codon found at nucleotide positions1340-1342 (FIG. 4; SEQ ID NO:9) [Kozak et al., supra]. The cDNA clonealso contains a polyadenylation sequence at its 3′ end. The predictedpolypeptide precursor is 417 amino acids long and has a calculatedmolecular weight of approximately 45 kDa and a PI of about 6.4.Hydropathy analysis (not shown) suggested the presence of a signalsequence (residues 1-24), followed by an extracellular domain (residues25-198), a transmembrane domain (residues 199-224), and an intracellulardomain (residues 225-417) (FIG. 4; SEQ ID NO:6). There are two potentialN-linked glycosylation sites at amino acid positions 67 and 106.

The ECD contains 4 cysteine-rich repeats which resemble thecorresponding regions of human TNFR1 (4 repeats), of human CD95 (3repeats) (FIG. 5) and of the other known TNFR family members (notshown). The ICD contains a death domain sequence that resembles thedeath domains found in the ICD of TNFR1 and CD95 and in cytoplasmicdeath signalling proteins such as human FADD/MORT1, TRADD, RIP, andDrosophila Reaper (FIG. 6). Both globally and in individual regions,Apo-3 is related more closely to TNFR1 than to CD95; the respectiveamino acid identities are 29.3% and 22.8% overall, 28.2% and 24.7% inthe ECD, 31.6% and 18.3% in the ICD, and 47.5% and 20% in the deathdomain.

The fetal lung cDNA clone, clone 5L8A.53, was identical to the fetalheart clone, with the following two exceptions: (1) it is 172 bp shorterat the 5′ region; and (2) it lacks the Ala residue at position 236,possibly due to differential mRNA splicing via two consecutive spliceacceptor consensus sites (FIG. 6).

As mentioned in Example 1 above, amino acids 1 to 181 of the Apo-2LIclone 18.1 shown in FIG. 1 (SEQ ID NO:1) are identical to amino acids 1to 181 of the Apo-3 polypeptide, shown in FIG. 4 (SEQ ID NO:6).

Example 5 Expression of Apo-3

A PRK5 mammalian expression plasmid (described in Example 2) carryingclone FH20A.57 (referred to in Example 4) was transfected transientlyinto HEK293 cells (referred to in the Examples above) by calciumphosphate precipitation and into HeLa-S3 cells (ATCC No. CCL 2.2) bystandard electroporation techniques.

Lysates of metabolically labeled transfected 293 cells were analyzed byimmunoprecipitation with a mouse antiserum raised against an Apo-2LI-IgGfusion protein. Transfected cells (5×10⁵ per lane) were labeledmetabolically by addition of 50 μCi ³⁵S-Met and ³⁵S-Cys to the growthmedia 24 hours after transfection. After a 6 hour incubation, the cellswere washed several times with PBS, lysed and subjected toimmunoprecipitation by anti-Apo-3 antiserum as described in Marsters etal., Proc. Natl. Acad. Sci., 92:5401-5405 (1995). The anti-Apo-3antiserum was raised in mice against a fusion protein containing theApo-2LI ECD (as described in Example 3).

A predominant radioactive band with a relative molecular weight of about47 kDa was observed in the pRK5-Apo-3-transfected cells, but not in thecells transfected with pRK5 alone (control) (See FIG. 8, lanes 1, 2).Given the potential glycosylation sites of Apo-3, the observed size isconsistent with the size of approximately 45 kDa predicted for the Apo-3polypeptide precursor.

Example 6 Apoptotic Activity of Apo-3

The transiently transfected HEK293 and HeLa cells described in Example 5were tested and analyzed for apoptotic activity 36 hours aftertransfection. Apoptosis was assessed morphologically or quantitated byFACS analysis of cells stained with fluoresceinisothiocyanate(FITC)-conjugated annexin V (Brand Applications) and propidium iodide(PI). The FACS analysis was conducted, using established criteria forapoptotic cell death, namely, the relation of fluorescence staining ofthe cells with two markers: (a) propidium iodide (PI) which stains theapoptotic cells but not the live cells, and (b) a fluorescent derivativeof the protein, annexin V, which binds to the exposed phosphatidylserinefound on the surface of apoptotic cells, but not on live cells[Darsynkiewicz et al., Methods in Cell. Biol., 41:15-38 (1994); Fadok etal., J. Immunol., 148:2207-2214 (1992); Koopman et al., Blood,84:1415-1420 (1994)]. The annexin V-positive/PI negative cells are inearly stages of apoptosis and double-positive cells are in lateapoptosis, while annexin V-negative/PI-positive cells are necrotic.Apoptosis was also assessed by DNA fragmentation testing.

Microscopic examination of the HEK 293 cells transfected with thepRK5-Apo-3 expression plasmid (see Example 5) showed a substantial lossof cell viability as compared to control cells transfected with pRK5alone; many of the Apo-3 transfected cells exhibited a characteristicapoptotic morphology of membrane blebbing and loss of cell volume (FIGS.9 a and b), suggesting cell death by apoptosis [Cohen, Advances inImmunology, 50:55-85 (1990)].

The FACS analysis also revealed that the Apo-3-transfected cells died byapoptosis, by virtue of the presence of exposed phosphatidylserine ontheir surface (FIGS. 9 e-i). It was found that the transienttransfection efficiency of the HEK 293 cells was 60-70%; therefore, totarget FACS analysis to cells that had taken up the plasmid DNA, the 293cells were co-transfected with a pRK5-CD4 expression vector (3 μg) as amarker and gated on CD4-positive cells (using phycoerythrin-conjugatedanti-CD4 antibody) for analysis. For the co-transfection, the totalamount of plasmid DNA was kept constant, but divided between differentplasmids. The Apo-3-transfected cells showed a marked increase in PI andannexin V-FITC staining as compared to pRK5-transfected control cellsindicating induction of apoptosis by Apo-3. (FIGS. 9 e and f).

The effect of the dose of plasmid on apoptosis was also tested in theFACS assay. (FIG. 9 i). Transfection of 293 cells with either Apo-3 orTNFR1 expression plasmids was associated with a dose-dependent increasein apoptosis; the effect of Apo-3 was more pronounced than that of TNFR1(FIG. 9 i). Similar results were obtained upon Apo-3 transfection of theHeLa cells (data not shown).

Apoptosis was also assayed by extraction of DNA from the cells, terminaltransferase-mediated ³²P-labelling of 3′ ends of DNA and 1.5% agarosegel electrophoresis as described by Moore et al., Cytotechnology,17:1-11 (1995). Analysis of the cellular DNA revealed that theApo-3-transfected cells showed a marked increase in DNA fragmentation ascompared to controls (FIG. 9 j, lanes 1, 2). The fragmented DNA migratedon agarose gels as a ladder of bands, indicating internucleosomal DNAcleavage, an indication of programmed cell death [Cohen, supra].

Example 7 Inhibition Assay Using CrmA

To investigate whether proteases such as ICE and CPP32/Yama play a rolein apoptosis-induction by Apo-3, an assay was conducted to determine ifCrmA inhibits Apo-3 function.

Co-transfection of HEK293 cells by a pRK5-CrmA expression plasmid (CrmAsequence reported in Ray et al., supra) and pRK5-Apo-3 did not affectthe apparent levels of Apo-3 expressed by the cells (FIG. 8, lane 3).CrmA, however, blocked Apo-3 associated apoptosis as analyzed bymorphological examination (FIGS. 9 c and d), FACS (FIGS. 9 g and h) andDNA fragmentation (FIG. 9 j, lanes 3,4) methods described in Example 6.A similar inhibitory effect of CrmA was observed in Apo-3-transfectedHeLa cells (data not shown).

CrmA, a poxvirus-derived inhibitor of the death proteases ICE andCPP32/Yama, blocks death signalling by TNFR1 and CD95. Accordingly, theassay results suggest that Apo-3, TNFR1 and CD95 engage a commonsignalling pathway to activate apoptotic cell death. In particular, theresults suggest that proteases such as ICE and CPP32/Yama may berequired for Apo-3 induced apoptosis.

Example 8 Activation of NF-κB by Apo-3

An assay was conducted to determine whether Apo-3 activates NF-κB.

HEK 293 cells were harvested 36 hours after transfection (see Example 5)and nuclear extracts were prepared and 1 μg of nuclear protein wasreacted with a ³²P_labelled NF-κB-specific synthetic oligonucleotideprobe ATCAGGGACTTTCCGCTGGGGACTTTCCG (SEQ ID NO: 10) [see, also, MacKayet al., J. Immunol., 153:5274-5284 (1994)], alone or together with a50-fold excess of unlabelled probe, or with an irrelevant ³²P-labelledsynthetic oligonucleotide AGGATGGGAAGTGTGTGATATATCCTTGAT (SEQ ID NO:11).DNA binding was analyzed by an electrophoretic mobility shift assay asdescribed by Hsu et al., supra; Marsters et al., supra, and MacKay etal., supra.

The results are shown in FIG. 10. The radioactive band at the bottom ofthe gel in all lanes is the free labelled probe, the two otherradioactive bands seen in lanes 1-3 represent non-specific interaction,as does the band common to lanes 1-3 and lanes 4-6. The top radioactiveband in lanes 4-6 represents the labelled NF-κB probe, whose migrationis delayed by specific interaction with activated NF-κB protein in thenuclear extracts.

Apo-3 transfected cells showed a significant increase in NF-κB-specificDNA binding activity relative to pRK5-transfected controls.TNFR1-transfected cells showed NF-κB activation as well; this activationappeared to be enhanced as compared to the Apo-3-transfected cells. Thedata thus shows that Apo-3 is capable of regulating transcription ofinflammatory response genes and in particular, may be linked to a NF-κBactivation pathway.

Example 9 Activation of JNK by Apo-3

An assay was conducted to determine whether Apo-3 activates c-JunN-terminal kinase (JNK). HEK 293 cells were harvested 36 hours aftertransfection (see Example 5) and JNK activation was determined byanalyzing phosphorylation of c-Jun with a SAPK/JNK assay kit (NewEngland Biolabs) according to manufacturer instructions.

Cell lysates were prepared from HEK 293 cells transfected with 10 μgpRK5, pRK5-TNFR1, or pRK5-Apo-3. JNK was precipitated with a GST-c-Junfusion protein bound to glutathione-sepharose beads. After washing, thekinase reaction was allowed to proceed in the presence of ATP, and wasresolved by SDS-PAGE. Phospho-c-Jun was detected by immunoblot withantibody specific for c-Jun phosphorylated on Ser63, a site importantfor transcriptional activity, using chemiluminescence detection. Theresults are shown in FIG. 11.

Apo-3 transfected cells showed a significant level of JNK activation ascompared to pRK5 transfected controls. TNFR1 transfected cells showedJNK activation as well; this activation appeared to be reduced relativeto that seen in Apo-3 transfected cells. The data thus shows that Apo-3is capable of regulating the stress-response signaling pathway which isalso known to be regulated by stimuli such as UV irradiation and variouscytokines.

Example 10 Northern Blot Analysis

Expression of Apo-3 mRNA in human tissues was examined by Northern blotanalysis. Human RNA blots were hybridized to a 206 bp ³²P-labelled DNAprobe based on the 3′ untranslated region of Apo-3; the probe wasgenerated by PCR with the 27 and 25 bp probes (described in Example 4)as PCR primers. Human fetal RNA blot MTN (Clontech) and human adult RNAblot MTN-II (Clontech) were incubated with the DNA probes. Blots wereincubated with the probes in hybridization buffer (5×SSPE; 2×Denhardt'ssolution; 100 mg/mL denatured sheared salmon sperm DNA; 50% formamide;2% SDS) for 60 hours at 42° C. The blots were washed several times in2×SSC; 0.05% SDS for 1 hour at room temperature, followed by a 30 minutewash in 0.1×SSC; 0.1% SDS at 50° C. The blots were developed afterovernight exposure.

As shown in FIG. 12, a predominant mRNA transcript of approximately 4 kbwas detected in adult spleen, thymus, and peripheral blood lymphocytes,and less abundantly in small intestine, colon, fetal lung, and fetalkidney. Additional transcripts of approximately 7 and 9 kb were seenmainly in fetal brain, lung and kidney, and in adult spleen and ovary.These results suggest that Apo-3 mRNA is expressed in several types oftissues, including both lymphoid and non-lymphoid tissues.

Example 11 Chromosomal Localization of the Apo-3 Gene

Chromosomal localization of the Apo-3 gene was examined by fluorescencein situ hybridization (“FISH”) to normal human lymphocyte chromosomes.

Initial testing by direct hybridization with the Apo-2LI (clone 18.1)cDNA (see Example 1 and FIG. 1) as a probe gave a relatively poor signalto background ratio (data not shown) but suggested that the gene islocated on chromosome 1p36. Further testing was conducted using theApo-3 cDNA probe and FISH mapping [as described by Lichter et al.,Science, 247:64-69 (1990)] of a human genomic p1-derived artificialchromosome (PAC) library (obtained from Dr. L. C. Tsui, University ofToronto, Toronto, Canada). The Apo-3 probes were biotinylated anddetected with avidin-FITC. The normal human lymphocyte chromosomes werecounterstained with PI and DAPI [Heng and Tsui, Chromosome, 102:325-332(1993)]. In addition to the “direct” FISH using the Apo-3 cDNA as aprobe, the probe was used to identify clones in the genomic PAC librarythat contain the Apo-3 gene, and the PACs were used as confirmatoryprobes in FISH. The regional assignment of the genomic probe wasdetermined by the analysis of 20 well-spread metaphases.

A positive PAC clone was mapped by FISH to the short arm of chromosome1, at position 1p36.3. A second Apo-3-positive genomic PAC was mapped tothe same position (data not shown). Positive hybridization signals at1p36.3 were noted at >95% of the cells. Signals were seen in bothchromosome 1 homologues in >90% of the positive spreads.

Recent reports disclose that a genomic region which is deleted incertain human neuroblastomas maps within 1p36.2-1p36.3, indicating thata tumor suppressor gene may be present at this locus. Four additionalTNFR gene family members, TNFR2, CD30, 4.1BB and OX40, reside in 1p36[see Gruss and Dower, supra] but are outside the deleted region [Whiteet al., Proc. Natl. Acad. Sci., 92:5520-5524 (1995)].

DEPOSIT OF MATERIAL

The following materials have been deposited with the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md., USA (ATCC):

Material ATCC Dep. No. Deposit Date Apo-2LI clone 18.1 97493 Mar. 27,1996 Apo-3 clone FH20.57 55820 Sep. 5, 1996

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC §122 and the Commissioner's rules pursuantthereto (including 37 CFR §1.14 with particular reference to 8860G 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. Isolated biologically active Apo-2LI having at least about 80%sequence identity with native sequence Apo-2LI having amino acidresidues 1 to 181 of SEQ ID NO:1.
 2. The Apo-2LI of claim 1 wherein saidApo-2LI has at least about 90% sequence identity.
 3. The Apo-2LI ofclaim 2 wherein said Apo-2LI has at least about 95% sequence identity.4. Isolated Apo-2LI comprising amino acid residues 1 to 181 of SEQ IDNO:1.
 5. A chimeric molecule comprising the Apo-2LI of claim 1 or claim4 fused to a heterologous amino acid sequence.
 6. The chimeric moleculeof claim 5 wherein said heterologous amino acid sequence is an epitopetag sequence.
 7. The chimeric molecule of claim 5 wherein saidheterologous amino acid sequence is an immunoglobulin sequence.
 8. Thechimeric molecule of claim 7 wherein said immunoglobulin sequence is anIgG.
 9. A dimer molecule comprising a first Apo-2LI and a secondApo-2LI.
 10. An antibody which binds to Apo-2LI.
 11. The antibody ofclaim 10 wherein said antibody is a monoclonal antibody.
 12. Isolatednucleic acid encoding Apo-2LI.
 13. The nucleic acid of claim 12 whereinsaid nucleic acid encodes an Apo-2LI comprising amino acid residues 1 to181 of SEQ ID NO:1.
 14. A vector comprising the nucleic acid of claim12.
 15. A host cell comprising the vector of claim
 14. 16. A method ofproducing Apo-2LI comprising culturing the host cell of claim 15 andrecovering the Apo-2LI from the host cell culture.
 17. An article ofmanufacture, comprising: a container; a label on said container; and acomposition contained within said container, said composition comprisingApo-2LI.
 18. The article of manufacture of claim 17 further comprisinginstructions for using the Apo-2LI in vivo or ex vivo.
 19. Isolatedbiologically active Apo-3 polypeptide having at least about 80% sequenceidentity with native sequence Apo-3 having amino acid residues 1 to 417of SEQ ID NO:6.
 20. The Apo-3 of claim 19 wherein said Apo-3 has atleast about 90% sequence identity.
 21. The Apo-3 of claim 20 whereinsaid Apo-3 has at least about 95% sequence identity.
 22. Isolated nativesequence Apo-3 comprising amino acid residues 1 to 417 of SEQ ID NO:6.23. Isolated biologically active polypeptide having at least about 80%sequence identity with the extracellular domain sequence of Apo-3 havingamino acid residues 1 to 198 of SEQ ID NO:6.
 24. The polypeptide ofclaim 23 wherein said polypeptide has at least about 90% sequenceidentity.
 25. The polypeptide of claim 24 wherein said polypeptide isApo-2LI.
 26. Isolated extracellular domain sequence of Apo-3 comprisingamino acid residues 1 to 198 of SEQ ID NO:6.
 27. Isolated death domainsequence of Apo-3 comprising amino acid residues 338 to 417 of SEQ IDNO:6.
 28. A chimeric molecule comprising the Apo-3 of claim 22 or theextracellular domain sequence of claim 23 fused to a heterologous aminoacid sequence.
 29. The chimeric molecule of claim 28 wherein saidheterologous amino acid sequence is an epitope tag sequence.
 30. Thechimeric molecule of claim 28 wherein said heterologous amino acidsequence is an immunoglobulin sequence.
 31. The chimeric molecule ofclaim 30 wherein said immunoglobulin sequence is an IgG.
 32. An antibodywhich binds to Apo-3 or to the extracellular domain sequence of claim23.
 33. The antibody of claim 32 wherein said antibody is a monoclonalantibody.
 34. Isolated nucleic acid encoding the Apo-3 of claim 22, theextracellular domain sequence of claim 23 or the death domain sequenceof claim
 27. 35. The nucleic acid of claim 34 wherein said nucleic acidencodes native sequence Apo-3 comprising amino acid residues 1 to 417 ofSEQ ID NO:6.
 36. A vector comprising the nucleic acid of claim
 34. 37.The vector of claim 36 operably linked to control sequences recognizedby a host cell transformed with the vector.
 38. A host cell comprisingthe vector of claim
 36. 39. A process of using a nucleic acid moleculeencoding Apo-3 to effect production of Apo-3 comprising culturing thehost cell of claim
 38. 40. A non-human, transgenic animal which containscells that express nucleic acid encoding Apo-3.
 41. The animal of claim40 which is a mouse or rat.
 42. A non-human, knockout animal whichcontains cells having an altered gene encoding Apo-3.
 43. The animal ofclaim 42 which is a mouse or rat.
 44. An article of manufacture,comprising a container and a composition contained within saidcontainer, wherein the composition includes Apo-3 polypeptide or Apo-3antibodies.
 45. The article of manufacture of claim 44 furthercomprising instructions for using the Apo-3 polypeptide or antibodies invivo or ex vivo.